Investigator, Howard Hughes Medical Institute at UCLA
| 2005–2011 | Assistant Professor of Biochemistry, University of Washington, Seattle |
| 2009–2011 | Howard Hughes Medical Institute Early Career Scientist |
| 2011 | Associate Professor of Biochemistry (Tenured), University of Washington |
| 2011–present | Affiliate Professor of Biochemistry, University of Washington, Seattle, WA |
| 2011–2017 | Group Leader, HHMI, Janelia Research Campus, Ashburn VA |
| 2017–Present | Investigator, Howard Hughes Medical Institute. |
| 2017–present | Professor of Biological Chemistry and Physiology, David Geffen Medical School. UCLA, CA. |
Honors
| 1996 | Deans List – Organic Chemistry, University of Auckland, New Zealand |
| 1996 | Deans List – Inorganic Chemistry, University of Auckland, New Zealand |
| 1997 | Center for Gene Technology Research Scholarship, University of Auckland, New Zealand |
| 1997 | Deans List – Inorganic Chemistry, University of Auckland, New Zealand |
| 1998 | Senior Prize in Biological Sciences, University of Auckland, New Zealand |
| 1998 | First Class Honors in Biological Sciences, University of Auckland, New Zealand |
| 1999 | University of Auckland Doctoral Scholarship, University of Auckland, New Zealand |
| 2000 | Contestable Travel Fund Award, University of Auckland, New Zealand |
| 2001 | Contestable Travel Fund Award, University of Auckland, New Zealand |
| 2009 | American Diabetes Association Career Development Award |
| 2009 | Howard Hughes Medical Institute Early Career Scientist |
| 2010 | New Investigator Science in Medicine Lecture |
| 2012 | Member, The Royal Society of New Zealand |
| 2017 | Investigator, Howard Hughes Medical Institute |
| 2018 | Chair elect, Biophysical Society cryoEM subgroup |
Publications
2019 |
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Dick, Markus; Sarai, Nicholas S; Martynowycz, Michael W; Gonen, Tamir; Arnold, Frances H Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation Journal Article J. Am. Chem. Soc., 2019. @article{pmid31747522, title = {Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation}, author = {Markus Dick and Nicholas S Sarai and Michael W Martynowycz and Tamir Gonen and Frances H Arnold}, doi = {10.1021/jacs.9b09864}, year = {2019}, date = {2019-11-01}, journal = {J. Am. Chem. Soc.}, abstract = {We previously engineered the tryptophan synthase β-subunit (TrpB), which catalyzes the condensation of L-serine and indole to L-tryptophan, to synthesize a range of noncanonical amino acids from L-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds by attacking N1 of the substrate. Protecting N1 encouraged evolution towards C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pfquat with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereo-center at the β-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the β-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pfquat offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We previously engineered the tryptophan synthase β-subunit (TrpB), which catalyzes the condensation of L-serine and indole to L-tryptophan, to synthesize a range of noncanonical amino acids from L-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds by attacking N1 of the substrate. Protecting N1 encouraged evolution towards C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pfquat with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereo-center at the β-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the β-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pfquat offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters. | |
Martynowycz, Michael W; Zhao, Wei; Hattne, Johan; Jensen, Grant J; Gonen, Tamir Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED Journal Article Structure, 27 (10), pp. 1594–1600, 2019. @article{pmid31422911, title = {Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED}, author = {Michael W Martynowycz and Wei Zhao and Johan Hattne and Grant J Jensen and Tamir Gonen}, doi = {10.1016/j.str.2019.07.004}, year = {2019}, date = {2019-10-01}, journal = {Structure}, volume = {27}, number = {10}, pages = {1594--1600}, abstract = {Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED. | |
Hattne, Johan; Martynowycz, Michael W; Penczek, Pawel A; Gonen, Tamir MicroED with the Falcon III direct electron detector Journal Article IUCrJ, 6 (Pt 5), pp. 921–926, 2019. @article{pmid31576224, title = {MicroED with the Falcon III direct electron detector}, author = {Johan Hattne and Michael W Martynowycz and Pawel A Penczek and Tamir Gonen}, doi = {10.1107/S2052252519010583}, year = {2019}, date = {2019-09-01}, journal = {IUCrJ}, volume = {6}, number = {Pt 5}, pages = {921--926}, abstract = {Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements. | |
Zhu, Lan ; Bu, Guanhong ; Shi, Dan ; Gonen, Tamir ; Liu Wei, Nannenga Brent L Structure determination from lipidic cubic phase embedded microcrystals by MicroED Online bioRxiv 2019. @online{2019_zhu, title = {Structure determination from lipidic cubic phase embedded microcrystals by MicroED}, author = {Zhu, Lan and Bu, Guanhong and Shi, Dan and Gonen, Tamir and Liu, Wei, Nannenga, Brent L.}, doi = {10.1101/724575}, year = {2019}, date = {2019-08-04}, organization = {bioRxiv}, abstract = {The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. Because crystals grown in LCP can be limited in size, improved techniques for structure determination from these small crystals are important. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-TEM to collect electron diffraction data and determine high-resolution structures from very thin micro and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP. Proteinase K in LCP was used as a model system, and several LCP sample preparation strategies were tested. Among these, treatment with 2-Methyl-2,4-pentanediol (MPD) and lipase were both able to reduce the viscosity of the LCP and produce quality cryo-EM grids with well-diffracting crystals. These results set the stage for the use of MicroED to analyze other microcrystalline samples grown in LCP.}, keywords = {}, pubstate = {published}, tppubtype = {online} } The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. Because crystals grown in LCP can be limited in size, improved techniques for structure determination from these small crystals are important. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-TEM to collect electron diffraction data and determine high-resolution structures from very thin micro and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP. Proteinase K in LCP was used as a model system, and several LCP sample preparation strategies were tested. Among these, treatment with 2-Methyl-2,4-pentanediol (MPD) and lipase were both able to reduce the viscosity of the LCP and produce quality cryo-EM grids with well-diffracting crystals. These results set the stage for the use of MicroED to analyze other microcrystalline samples grown in LCP. | |
Warmack, Rebeccah A; Boyer, David R; Zee, Chih-Te; Richards, Logan S; Sawaya, Michael R; Cascio, Duilio; Gonen, Tamir; Eisenberg, David S; Clarke, Steven G Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface Journal Article Nat Commun, 10 (1), pp. 3357, 2019. @article{pmid31350392, title = {Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface}, author = {Rebeccah A Warmack and David R Boyer and Chih-Te Zee and Logan S Richards and Michael R Sawaya and Duilio Cascio and Tamir Gonen and David S Eisenberg and Steven G Clarke}, doi = {10.1038/s41467-019-11183-z}, year = {2019}, date = {2019-07-26}, journal = {Nat Commun}, volume = {10}, number = {1}, pages = {3357}, abstract = {Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD. | |
Ting, Chi P; Funk, Michael A; Halaby, Steve L; Zhang, Zhengan; Gonen, Tamir; van der Donk, Wilfred A Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products Journal Article Science, 365 (6450), pp. 280–284, 2019. @article{pmid31320540, title = {Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products}, author = {Chi P Ting and Michael A Funk and Steve L Halaby and Zhengan Zhang and Tamir Gonen and Wilfred A van der Donk}, doi = {10.1126/science.aau6232}, year = {2019}, date = {2019-07-19}, journal = {Science}, volume = {365}, number = {6450}, pages = {280--284}, abstract = {Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA-dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid-derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid-derived natural products.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA-dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid-derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid-derived natural products. | |
Martynowycz, Michael W; Gonen, Tamir MicroED Structure of Hexagonal Ice Ih Online ChemRxiv 2019. @online{2019_martynowycz, title = {MicroED Structure of Hexagonal Ice Ih}, author = {Martynowycz, Michael W. and Gonen, Tamir}, doi = {10.26434/chemrxiv.8298641.v1}, year = {2019}, date = {2019-06-20}, organization = {ChemRxiv}, abstract = {The structure of ice Ih is solved from a single nanocrystal to a resolution of 0.53Å using the cryoEM method microcrystal electron diffraction (MicroED). Data were collected at just above liquid nitrogen temperatures (~80K) in ultra-high vacuum (~8 x 10-7 Pa) using a total exposure of less than 1e- Å-2. The model has the same unit cell dimensions and space group as structures previously determined by both X-ray and neutron scattering of ice Ih, and obeys the Bernal–Fowler ice rules. Both axial and distal hydrogen densities between oxygen atoms of non-deuterated water ice are resolved. Unaccounted for density between axial hydrogen atoms is observed, and may be a direct observation of a polar bond caused by the electric dipole between oxygen and hydrogen atoms. These observations may have implications for the effects of electron radiation on non-terrestrial ice formations because the conditions experienced by the sample in these experiments are mimetic to those found in near solar space or some planetary bodies without atmospheres, where water ice deposits are exposed to high-energy cosmic rays, thermal stellar emissions, and radiation stemming from solar flares.}, keywords = {}, pubstate = {published}, tppubtype = {online} } The structure of ice Ih is solved from a single nanocrystal to a resolution of 0.53Å using the cryoEM method microcrystal electron diffraction (MicroED). Data were collected at just above liquid nitrogen temperatures (~80K) in ultra-high vacuum (~8 x 10-7 Pa) using a total exposure of less than 1e- Å-2. The model has the same unit cell dimensions and space group as structures previously determined by both X-ray and neutron scattering of ice Ih, and obeys the Bernal–Fowler ice rules. Both axial and distal hydrogen densities between oxygen atoms of non-deuterated water ice are resolved. Unaccounted for density between axial hydrogen atoms is observed, and may be a direct observation of a polar bond caused by the electric dipole between oxygen and hydrogen atoms. These observations may have implications for the effects of electron radiation on non-terrestrial ice formations because the conditions experienced by the sample in these experiments are mimetic to those found in near solar space or some planetary bodies without atmospheres, where water ice deposits are exposed to high-energy cosmic rays, thermal stellar emissions, and radiation stemming from solar flares. | |
de la Cruz, Jason M; Martynowycz, Michael W; Hattne, Johan; Gonen, Tamir MicroED data collection with SerialEM Journal Article Ultramicroscopy, 201 , pp. 77–80, 2019. @article{pmid30986656, title = {MicroED data collection with SerialEM}, author = {Jason M de la Cruz and Michael W Martynowycz and Johan Hattne and Tamir Gonen}, doi = {10.1016/j.ultramic.2019.03.009}, year = {2019}, date = {2019-06-01}, journal = {Ultramicroscopy}, volume = {201}, pages = {77--80}, abstract = {The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses. | |
Nannenga, Brent L; Gonen, Tamir The cryo-EM method microcrystal electron diffraction (MicroED) Journal Article Nat. Methods, 16 (5), pp. 369–379, 2019. @article{pmid31040436, title = {The cryo-EM method microcrystal electron diffraction (MicroED)}, author = {Brent L Nannenga and Tamir Gonen}, doi = {10.1038/s41592-019-0395-x}, year = {2019}, date = {2019-04-29}, journal = {Nat. Methods}, volume = {16}, number = {5}, pages = {369--379}, abstract = {In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users. | |
Martynowycz, Michael W; Zhao, Wei; Hattne, Johan; Jensen, Grant J; Gonen, Tamir Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size Journal Article Structure, 27 (3), pp. 545–548, 2019. @article{pmid30661853, title = {Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size}, author = {Michael W Martynowycz and Wei Zhao and Johan Hattne and Grant J Jensen and Tamir Gonen}, doi = {10.1016/j.str.2018.12.003}, year = {2019}, date = {2019-03-05}, journal = {Structure}, volume = {27}, number = {3}, pages = {545--548}, abstract = {Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED. | |
Zee, Chih-Te; Glynn, Calina; Gallagher-Jones, Marcus; Miao, Jennifer; Santiago, Carlos G; Cascio, Duilio; Gonen, Tamir; Sawaya, Michael R; Rodriguez, Jose A Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ Journal Article IUCrJ, 6 (Pt 2), pp. 197–205, 2019. @article{pmid30867917, title = {Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ}, author = {Chih-Te Zee and Calina Glynn and Marcus Gallagher-Jones and Jennifer Miao and Carlos G Santiago and Duilio Cascio and Tamir Gonen and Michael R Sawaya and Jose A Rodriguez}, doi = {10.1107/S2052252518017621}, year = {2019}, date = {2019-03-01}, journal = {IUCrJ}, volume = {6}, number = {Pt 2}, pages = {197--205}, abstract = {The ice-nucleation protein InaZ from Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. Ab initio structures of this repeat segment, resolved to 1.1 Ã… by microfocus X-ray crystallography and to 0.9 Ã… by the cryo-EM method MicroED, were determined from both racemic and homochiral crystals. The benefits of racemic protein crystals for structure determination by MicroED were evaluated and it was confirmed that the phase restriction introduced by crystal centrosymmetry increases the number of successful trials during the ab initio phasing of the electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel β-sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all-left-handed sheets mate with their all-right-handed counterparts. This determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with submicrometre-sized crystals.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The ice-nucleation protein InaZ from Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. Ab initio structures of this repeat segment, resolved to 1.1 Ã… by microfocus X-ray crystallography and to 0.9 Ã… by the cryo-EM method MicroED, were determined from both racemic and homochiral crystals. The benefits of racemic protein crystals for structure determination by MicroED were evaluated and it was confirmed that the phase restriction introduced by crystal centrosymmetry increases the number of successful trials during the ab initio phasing of the electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel β-sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all-left-handed sheets mate with their all-right-handed counterparts. This determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with submicrometre-sized crystals. | |
Ma, Jinming; Lei, Hsiang-Ting; Reyes, Francis E; Sanchez-Martinez, Silvia; Sarhan, Maen F; Hattne, Johan; Gonen, Tamir Structural basis for substrate binding and specificity of a sodium-alanine symporter AgcS Journal Article Proc. Natl. Acad. Sci. U.S.A., 116 (6), pp. 2086–2090, 2019. @article{pmid30659158, title = {Structural basis for substrate binding and specificity of a sodium-alanine symporter AgcS}, author = {Jinming Ma and Hsiang-Ting Lei and Francis E Reyes and Silvia Sanchez-Martinez and Maen F Sarhan and Johan Hattne and Tamir Gonen}, doi = {10.1073/pnas.1806206116}, year = {2019}, date = {2019-01-18}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {116}, number = {6}, pages = {2086--2090}, abstract = {The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters. | |
Griner, Sarah L; Seidler, Paul; Bowler, Jeannette; Murray, Kevin A; Yang, Tianxiao Peter; Sahay, Shruti; Sawaya, Michael R; Cascio, Duilio; Rodriguez, Jose A; Philipp, Stephan; Sosna, Justyna; Glabe, Charles G; Gonen, Tamir; Eisenberg, David S Structure-based inhibitors of amyloid beta core suggest a common interface with tau Journal Article Elife, 8 , 2019. @article{pmid31612856, title = {Structure-based inhibitors of amyloid beta core suggest a common interface with tau}, author = {Sarah L Griner and Paul Seidler and Jeannette Bowler and Kevin A Murray and Tianxiao Peter Yang and Shruti Sahay and Michael R Sawaya and Duilio Cascio and Jose A Rodriguez and Stephan Philipp and Justyna Sosna and Charles G Glabe and Tamir Gonen and David S Eisenberg}, doi = {10.7554/eLife.46924.001}, year = {2019}, date = {2019-01-01}, journal = {Elife}, volume = {8}, abstract = {Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline. | |
2018 |
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Purdy, Michael D; Shi, Dan; Chrustowicz, Jakub; Hattne, Johan; Gonen, Tamir; Yeager, Mark MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat Journal Article Proc. Natl. Acad. Sci. U.S.A., 115 (52), pp. 13258–13263, 2018. @article{pmid30530702, title = {MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat}, author = {Michael D Purdy and Dan Shi and Jakub Chrustowicz and Johan Hattne and Tamir Gonen and Mark Yeager}, doi = {10.1073/pnas.1806806115}, year = {2018}, date = {2018-12-26}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {115}, number = {52}, pages = {13258--13263}, abstract = {HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.}, keywords = {}, pubstate = {published}, tppubtype = {article} } HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design. | |
Jones, Christopher G; Martynowycz, Michael W; Hattne, Johan; Fulton, Tyler J; Stoltz, Brian M; Rodriguez, Jose A; Nelson, Hosea M; Gonen, Tamir The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination Journal Article ACS Cent Sci, 4 (11), pp. 1587–1592, 2018. @article{pmid30555912, title = {The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination}, author = {Christopher G Jones and Michael W Martynowycz and Johan Hattne and Tyler J Fulton and Brian M Stoltz and Jose A Rodriguez and Hosea M Nelson and Tamir Gonen}, doi = {10.1021/acscentsci.8b00760}, year = {2018}, date = {2018-11-02}, journal = {ACS Cent Sci}, volume = {4}, number = {11}, pages = {1587--1592}, abstract = {In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule's structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10-15 g) resulting in atomic resolution (<1 Ã…) crystal structures in minutes.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule's structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10-15 g) resulting in atomic resolution (<1 Ã…) crystal structures in minutes. | |
Martynowycz, Michael W; Zhao, Wei ; Hattne, Johen ; Jensen, Grant J; Gonen, Tamir Collection of continuous rotation MicroED Data from Ion Beam Milled Crystals of Any Size Online bioRxiv 2018. @online{2018_martynowycz, title = {Collection of continuous rotation MicroED Data from Ion Beam Milled Crystals of Any Size}, author = {Martynowycz, Michael W. and Zhao, Wei and Hattne, Johen and Jensen, Grant J. and Gonen, Tamir}, doi = {10.1101/425611}, year = {2018}, date = {2018-09-24}, organization = {bioRxiv}, abstract = {Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoEM as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to ideal dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.}, keywords = {}, pubstate = {published}, tppubtype = {online} } Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoEM as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to ideal dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED. | |
Ma, Jinming ; Lei, Hsiang-Ting ; Gonen, Tamir A conformational change in the N terminus of SLC38A9 signals mTORC1 activation Online bioRxiv 2018. @online{2018_ma_b, title = {A conformational change in the N terminus of SLC38A9 signals mTORC1 activation}, author = {Ma, Jinming and Lei, Hsiang-Ting and Gonen, Tamir}, doi = {10.1101/339937}, year = {2018}, date = {2018-06-06}, journal = {bio}, organization = {bioRxiv}, abstract = {mTORC1 is a central signal hub that integrates multiple environmental cues, such as cellular stresses, energy levels, nutrients and certain amino acids, to modulate metabolic status and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, has emerged as a sensor for luminal arginine levels and as an activator of mTOCRC1. The activation of mTORC1 occurs through the N-terminal domain of SLC38A9. Here, we determined the crystal structure of SLC38A9 and surprisingly found its N-terminal fragment inserted deep into the transporter, bound in the substrate binding pocket where normally arginine would bind. Compared with our recent arginine bound structure of SLC38A9, a significant conformational change of the N-terminal domain was observed. A ball-and-chain model is proposed for mTORC1 activation where in the starved state the N-terminal domain of SLC38A9 is buried deep in the transporter but in the fed state the N-terminal domain could be released becoming free to bind the Rag GTPase complex and to activate mTORC1. This work provides important new insights into how SLC38A9 senses the fed state and activates the mTORC1 pathways in response to dietary amino acids. }, keywords = {}, pubstate = {published}, tppubtype = {online} } mTORC1 is a central signal hub that integrates multiple environmental cues, such as cellular stresses, energy levels, nutrients and certain amino acids, to modulate metabolic status and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, has emerged as a sensor for luminal arginine levels and as an activator of mTOCRC1. The activation of mTORC1 occurs through the N-terminal domain of SLC38A9. Here, we determined the crystal structure of SLC38A9 and surprisingly found its N-terminal fragment inserted deep into the transporter, bound in the substrate binding pocket where normally arginine would bind. Compared with our recent arginine bound structure of SLC38A9, a significant conformational change of the N-terminal domain was observed. A ball-and-chain model is proposed for mTORC1 activation where in the starved state the N-terminal domain of SLC38A9 is buried deep in the transporter but in the fed state the N-terminal domain could be released becoming free to bind the Rag GTPase complex and to activate mTORC1. This work provides important new insights into how SLC38A9 senses the fed state and activates the mTORC1 pathways in response to dietary amino acids. | |
Lei, Hsiang-Ting; Ma, Jinming; Martinez, Silvia Sanchez; Gonen, Tamir Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state Journal Article Nat. Struct. Mol. Biol., 25 (6), pp. 522–527, 2018. @article{pmid29872228, title = {Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state}, author = {Hsiang-Ting Lei and Jinming Ma and Silvia Sanchez Martinez and Tamir Gonen}, doi = {10.1038/s41594-018-0072-2}, year = {2018}, date = {2018-06-05}, journal = {Nat. Struct. Mol. Biol.}, volume = {25}, number = {6}, pages = {522--527}, abstract = {Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters. | |
Liu, Shian; Gonen, Tamir MicroED structure of the NaK ion channel reveals a Na⁺ partition process into the selectivity filter Journal Article Commun Biol, 1 , pp. 38, 2018. @article{pmid30167468, title = {MicroED structure of the NaK ion channel reveals a Na⁺ partition process into the selectivity filter}, author = {Shian Liu and Tamir Gonen}, doi = {10.1038/s42003-018-0040-8}, year = {2018}, date = {2018-05-03}, journal = {Commun Biol}, volume = {1}, pages = {38}, abstract = {Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics. | |
Hattne, Johan; Shi, Dan; Glynn, Calina; Zee, Chih-Te; Gallagher-Jones, Marcus; Martynowycz, Michael W; Rodriguez, Jose A; Gonen, Tamir Analysis of Global and Site-Specific Radiation Damage in Cryo-EM Journal Article Structure, 26 (5), pp. 759–766, 2018. @article{pmid29706530, title = {Analysis of Global and Site-Specific Radiation Damage in Cryo-EM}, author = {Johan Hattne and Dan Shi and Calina Glynn and Chih-Te Zee and Marcus Gallagher-Jones and Michael W Martynowycz and Jose A Rodriguez and Tamir Gonen}, doi = {10.1016/j.str.2018.03.021}, year = {2018}, date = {2018-05-01}, journal = {Structure}, volume = {26}, number = {5}, pages = {759--766}, abstract = {Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data. | |
Ma, Jinming ; Lei, Hsiang-Ting ; Reyes, Francis E; Sanchez-Martinez, Silvia ; Sarhan, Maen ; Gonen, Tamir Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS Online bioRxiv 2018. @online{2018_ma, title = {Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS}, author = {Ma, Jinming and Lei, Hsiang-Ting and Reyes, Francis E. and Sanchez-Martinez, Silvia and Sarhan, Maen and Gonen, Tamir}, doi = {10.1101/293811}, year = {2018}, date = {2018-04-03}, organization = {bioRxiv}, abstract = {The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.}, keywords = {}, pubstate = {published}, tppubtype = {online} } The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters. | |
Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Yeates, Todd O Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system Journal Article Proc. Natl. Acad. Sci. U.S.A., 115 (13), pp. 3362–3367, 2018. @article{pmid29507202, title = {Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system}, author = {Yuxi Liu and Shane Gonen and Tamir Gonen and Todd O Yeates}, doi = {10.1073/pnas.1718825115}, year = {2018}, date = {2018-03-27}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {115}, number = {13}, pages = {3362--3367}, abstract = {Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell. | |
Guenther, Elizabeth L; Ge, Peng; Trinh, Hamilton; Sawaya, Michael R; Cascio, Duilio; Boyer, David R; Gonen, Tamir; Zhou, Hong Z; Eisenberg, David S Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2 Journal Article Nat. Struct. Mol. Biol., 25 (4), pp. 311–319, 2018. @article{pmid29531287, title = {Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2}, author = {Elizabeth L Guenther and Peng Ge and Hamilton Trinh and Michael R Sawaya and Duilio Cascio and David R Boyer and Tamir Gonen and Hong Z Zhou and David S Eisenberg}, doi = {10.1038/s41594-018-0045-5}, year = {2018}, date = {2018-03-12}, journal = {Nat. Struct. Mol. Biol.}, volume = {25}, number = {4}, pages = {311--319}, abstract = {Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures. | |
Martynowycz, Michael W; Gonen, Tamir From electron crystallography of 2D crystals to MicroED of 3D crystals Journal Article Curr Opin Colloid Interface Sci, 34 , pp. 9–16, 2018. @article{pmid30166936, title = {From electron crystallography of 2D crystals to MicroED of 3D crystals}, author = {Michael W Martynowycz and Tamir Gonen}, doi = {10.1016/j.cocis.2018.01.010}, year = {2018}, date = {2018-03-01}, journal = {Curr Opin Colloid Interface Sci}, volume = {34}, pages = {9--16}, abstract = {Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields. | |
Hughes, Michael P; Sawaya, Michael R; Boyer, David R; Goldschmidt, Lukasz; Rodriguez, Jose A; Cascio, Duilio; Chong, Lisa; Gonen, Tamir; Eisenberg, David S Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks Journal Article Science, 359 (6376), pp. 698–701, 2018. @article{pmid29439243, title = {Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks}, author = {Michael P Hughes and Michael R Sawaya and David R Boyer and Lukasz Goldschmidt and Jose A Rodriguez and Duilio Cascio and Lisa Chong and Tamir Gonen and David S Eisenberg}, doi = {10.1126/science.aan6398}, year = {2018}, date = {2018-02-09}, journal = {Science}, volume = {359}, number = {6376}, pages = {698--701}, abstract = {Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies. | |
Nannenga, Brent L; Gonen, Tamir MicroED: a versatile cryoEM method for structure determination Journal Article Emerg Top Life Sci, 2 (1), pp. 1–8, 2018. @article{pmid30167465, title = {MicroED: a versatile cryoEM method for structure determination}, author = {Brent L Nannenga and Tamir Gonen}, doi = {10.1042/ETLS20170082}, year = {2018}, date = {2018-02-06}, journal = {Emerg Top Life Sci}, volume = {2}, number = {1}, pages = {1--8}, abstract = {Micro-electron diffraction, or MicroED, is a structure determination method that uses a cryo-transmission electron microscope to collect electron diffraction data from nanocrystals. This technique has been successfully used to determine the high-resolution structures of many targets from crystals orders of magnitude smaller than what is needed for X-ray diffraction experiments. In this review, we will describe the MicroED method and recent structures that have been determined. Additionally, applications of electron diffraction to the fields of small molecule crystallography and materials science will be discussed.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Micro-electron diffraction, or MicroED, is a structure determination method that uses a cryo-transmission electron microscope to collect electron diffraction data from nanocrystals. This technique has been successfully used to determine the high-resolution structures of many targets from crystals orders of magnitude smaller than what is needed for X-ray diffraction experiments. In this review, we will describe the MicroED method and recent structures that have been determined. Additionally, applications of electron diffraction to the fields of small molecule crystallography and materials science will be discussed. | |
Gallagher-Jones, Marcus; Glynn, Calina; Boyer, David R; Martynowycz, Michael W; Hernandez, Evelyn; Miao, Jennifer; Zee, Chih-Te; Novikova, Irina V; Goldschmidt, Lukasz; McFarlane, Heather T; Helguera, Gustavo F; Evans, James E; Sawaya, Michael R; Cascio, Duilio; Eisenberg, David S; Gonen, Tamir; Rodriguez, Jose A Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp Journal Article Nat. Struct. Mol. Biol., 25 (2), pp. 131–134, 2018. @article{pmid29335561, title = {Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp}, author = {Marcus Gallagher-Jones and Calina Glynn and David R Boyer and Michael W Martynowycz and Evelyn Hernandez and Jennifer Miao and Chih-Te Zee and Irina V Novikova and Lukasz Goldschmidt and Heather T McFarlane and Gustavo F Helguera and James E Evans and Michael R Sawaya and Duilio Cascio and David S Eisenberg and Tamir Gonen and Jose A Rodriguez}, doi = {10.1038/s41594-017-0018-0}, year = {2018}, date = {2018-01-15}, journal = {Nat. Struct. Mol. Biol.}, volume = {25}, number = {2}, pages = {131--134}, abstract = {The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-Ã¥ngström-resolution structure of a protofibril formed by a wild-type segment from the β2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-Ã¥ngström-resolution structure of a protofibril formed by a wild-type segment from the β2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'. | |
2017 |
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Seidler, P M; Boyer, D R; Rodriguez, J A; Sawaya, M R; Cascio, D; Murray, K; Gonen, T; Eisenberg, D S Structure-based inhibitors of tau aggregation Journal Article Nat Chem, 10 (2), pp. 170–176, 2017. @article{pmid29359764, title = {Structure-based inhibitors of tau aggregation}, author = {P M Seidler and D R Boyer and J A Rodriguez and M R Sawaya and D Cascio and K Murray and T Gonen and D S Eisenberg}, doi = {10.1038/nchem.2889}, year = {2017}, date = {2017-11-20}, journal = {Nat Chem}, volume = {10}, number = {2}, pages = {170--176}, abstract = {Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau. | |
Vergara, Sandra; Lukes, Dylan A; Martynowycz, Michael W; Santiago, Ulises; Plascencia-Villa, Germán; Weiss, Simon C; de la Cruz, Jason M; Black, David M; Alvarez, Marcos M; López-Lozano, Xochitl; Barnes, Christopher O; Lin, Guowu; Weissker, Hans-Christian; Whetten, Robert L; Gonen, Tamir; Yacaman, Miguel Jose; Calero, Guillermo MicroED Structure of Au₁₄₆(p-MBA)₅₇ at Subatomic Resolution Reveals a Twinned FCC Cluster Journal Article J Phys Chem Lett, 8 (22), pp. 5523–5530, 2017. @article{pmid29072840, title = {MicroED Structure of Au₁₄₆(p-MBA)₅₇ at Subatomic Resolution Reveals a Twinned FCC Cluster}, author = {Sandra Vergara and Dylan A Lukes and Michael W Martynowycz and Ulises Santiago and Germán Plascencia-Villa and Simon C Weiss and Jason M de la Cruz and David M Black and Marcos M Alvarez and Xochitl López-Lozano and Christopher O Barnes and Guowu Lin and Hans-Christian Weissker and Robert L Whetten and Tamir Gonen and Miguel Jose Yacaman and Guillermo Calero}, url = {https://cryoem.ucla.edu/wp-content/uploads/2017_vargara.pdf}, doi = {10.1021/acs.jpclett.7b02621}, year = {2017}, date = {2017-10-26}, journal = {J Phys Chem Lett}, volume = {8}, number = {22}, pages = {5523--5530}, abstract = {Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure, whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure, whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault. | |
Rodriguez, Jose A; Eisenberg, David S; Gonen, Tamir Taking the measure of MicroED Journal Article Curr. Opin. Struct. Biol., 46 , pp. 79–86, 2017. @article{pmid28648726, title = {Taking the measure of MicroED}, author = {Jose A Rodriguez and David S Eisenberg and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2017_rodriguez.pdf, Main text}, doi = {10.1016/j.sbi.2017.06.004}, year = {2017}, date = {2017-10-01}, journal = {Curr. Opin. Struct. Biol.}, volume = {46}, pages = {79--86}, abstract = {It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.}, keywords = {}, pubstate = {published}, tppubtype = {article} } It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED. | |
Wright, Zoë V F; McCarthy, Stephen; Dickman, Rachel; Reyes, Francis E; Sanchez-Martinez, Silvia; Cryar, Adam; Kilford, Iian; Hall, Adrian; Takle, Andrew K; Topf, Maya; Gonen, Tamir; Thalassinos, Konstantinos; Tabor, Alethea B J. Am. Chem. Soc., 139 (37), pp. 13063–13075, 2017. @article{pmid28880078, title = {The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Naᵥ1.7}, author = {Zoë V F Wright and Stephen McCarthy and Rachel Dickman and Francis E Reyes and Silvia Sanchez-Martinez and Adam Cryar and Iian Kilford and Adrian Hall and Andrew K Takle and Maya Topf and Tamir Gonen and Konstantinos Thalassinos and Alethea B Tabor}, url = {https://cryoem.ucla.edu/wp-content/uploads/2017_wright.pdf, Main text}, doi = {10.1021/jacs.7b06506}, year = {2017}, date = {2017-09-07}, journal = {J. Am. Chem. Soc.}, volume = {139}, number = {37}, pages = {13063--13075}, abstract = {Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Naᵥ1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Naᵥ1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate. | |
Liu, Shian; Hattne, Johan; Reyes, Francis E; Sanchez-Martinez, Silvia; de la Cruz, Jason M; Shi, Dan; Gonen, Tamir Atomic resolution structure determination by the cryo-EM method MicroED Journal Article Protein Sci., 26 (1), pp. 8–15, 2017. @article{pmid27452773, title = {Atomic resolution structure determination by the cryo-EM method MicroED}, author = {Shian Liu and Johan Hattne and Francis E Reyes and Silvia Sanchez-Martinez and Jason M de la Cruz and Dan Shi and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2016_liu.pdf, Main text}, doi = {10.1002/pro.2989}, year = {2017}, date = {2017-07-25}, journal = {Protein Sci.}, volume = {26}, number = {1}, pages = {8--15}, abstract = {The electron cryo-microscopy (cryoEM) method MicroED has been rapidly developing. In this review we highlight some of the key steps in MicroED from crystal analysis to structure determination. We compare and contrast MicroED and the latest X-ray based diffraction method the X-ray free-electron laser (XFEL). Strengths and shortcomings of both MicroED and XFEL are discussed. Finally, all current MicroED structures are tabulated with a view to the future.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The electron cryo-microscopy (cryoEM) method MicroED has been rapidly developing. In this review we highlight some of the key steps in MicroED from crystal analysis to structure determination. We compare and contrast MicroED and the latest X-ray based diffraction method the X-ray free-electron laser (XFEL). Strengths and shortcomings of both MicroED and XFEL are discussed. Finally, all current MicroED structures are tabulated with a view to the future. | |
Hughes, Michael P; Sawaya, Michael R; Goldschidt, Lukasz ; Rodriguez Jose A. ad Cascio, Duilio ; Gonen, Tamir ; Eisenberg, David S Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets Online bioRxiv 2017. @online{2017_hughes, title = {Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets}, author = {Hughes, Michael P. and Sawaya, Michael R. and Goldschidt, Lukasz and Rodriguez, Jose A. ad Cascio, Duilio and Gonen, Tamir and Eisenberg, David S.}, doi = {10.1101/153817}, year = {2017}, date = {2017-06-22}, organization = {bioRxiv}, abstract = {Control of metabolism by compartmentation is a widespread feature of higher cells. Recent studies have focused on dynamic intracellular bodies such as stress granules, P-bodies, nucleoli, and metabolic puncta. These bodies appear as separate phases, some containing reversible, amyloid-like fibrils formed by interactions of low-complexity protein domains. Here we report five atomic structures of segments of low-complexity domains from granule-forming proteins, one determined to 1.1 Å resolution by micro-electron diffraction. Four of these interacting protein segments show common characteristics, all in contrast to pathogenic amyloid: kinked peptide backbones, small surface areas of interaction, and predominate attractions between aromatic side-chains. By computationally threading the human proteome on three of our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such reversible interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, keratins, and cornified envelope proteins, consistent with the capacity of cells to form a wide variety of dynamic intracellular bodies.}, keywords = {}, pubstate = {published}, tppubtype = {online} } Control of metabolism by compartmentation is a widespread feature of higher cells. Recent studies have focused on dynamic intracellular bodies such as stress granules, P-bodies, nucleoli, and metabolic puncta. These bodies appear as separate phases, some containing reversible, amyloid-like fibrils formed by interactions of low-complexity protein domains. Here we report five atomic structures of segments of low-complexity domains from granule-forming proteins, one determined to 1.1 Å resolution by micro-electron diffraction. Four of these interacting protein segments show common characteristics, all in contrast to pathogenic amyloid: kinked peptide backbones, small surface areas of interaction, and predominate attractions between aromatic side-chains. By computationally threading the human proteome on three of our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such reversible interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, keratins, and cornified envelope proteins, consistent with the capacity of cells to form a wide variety of dynamic intracellular bodies. | |
de la Cruz, Jason M; Martynowycz, Michael ; Hattne, Johan ; Shi, Dan ; Gonen, Tamir bioRxiv 2017. @online{2017_delacruz_c, title = {A method to minimize condenser lens-induced hysteresis effects in a JEOL JEM-3200FSC microscope to enable stable cryoEM low-dose operations}, author = {de la Cruz, M. Jason and Martynowycz, Michael and Hattne, Johan and Shi, Dan and Gonen, Tamir}, doi = {10.1101/153395}, year = {2017}, date = {2017-06-21}, organization = {bioRxiv}, abstract = {Low dose imaging procedures are key for a successful cryoEM experiment (whether by electron cryotomography, single particle analysis, electron crystallography, or MicroED). We present a method to minimize magnetic hysteresis of the condenser lens system in the JEOL JEM-3200FSC transmission electron microscope (TEM) in order to maintain a stable optical axis for the beam path of low-dose imaging. The simple procedure involves independent voltage ramping of the CL1 and CL2 lenses immediately before switching to the focusing and exposure beam settings for data collection.}, keywords = {}, pubstate = {published}, tppubtype = {online} } Low dose imaging procedures are key for a successful cryoEM experiment (whether by electron cryotomography, single particle analysis, electron crystallography, or MicroED). We present a method to minimize magnetic hysteresis of the condenser lens system in the JEOL JEM-3200FSC transmission electron microscope (TEM) in order to maintain a stable optical axis for the beam path of low-dose imaging. The simple procedure involves independent voltage ramping of the CL1 and CL2 lenses immediately before switching to the focusing and exposure beam settings for data collection. | |
Martynowycz, Michael W; Glynn, Calina ; Miao, Jennifer M; de la Cruz, Jason M; Hattne, Johan ; Shi, Dan ; Cascio, Duilio ; Jose, Rodriguez; Gonen, Tamir MicroED Structures from Micrometer Thick Protein Crystals Online bioRxiv 2017. @online{2017_martynowycz, title = {MicroED Structures from Micrometer Thick Protein Crystals}, author = {Martynowycz, Michael W. and Glynn, Calina and Miao, Jennifer M. and de la Cruz, M. Jason and Hattne, Johan and Shi, Dan and Cascio, Duilio and Rodriguez Jose and Gonen, Tamir}, doi = {10.1101/152504}, year = {2017}, date = {2017-06-20}, organization = {bioRxiv}, abstract = {Theoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments. Summary: Atomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude.}, keywords = {}, pubstate = {published}, tppubtype = {online} } Theoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments. Summary: Atomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude. | |
Krotee, Pascal; Griner, Sarah L; Sawaya, Michael R; Cascio, Duilio; Rodriguez, Jose A; Shi, Dan; Philipp, Stephan; Murray, Kevin; Saelices, Lorena; Lee, Ji; Seidler, Paul; Glabe, Charles G; Jiang, Lin; Gonen, Tamir; Eisenberg, David S Common fibrillar spines of amyloid-β and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors Journal Article J. Biol. Chem., 293 (8), pp. 2888–2902, 2017. @article{pmid29282295, title = {Common fibrillar spines of amyloid-β and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors}, author = {Pascal Krotee and Sarah L Griner and Michael R Sawaya and Duilio Cascio and Jose A Rodriguez and Dan Shi and Stephan Philipp and Kevin Murray and Lorena Saelices and Ji Lee and Paul Seidler and Charles G Glabe and Lin Jiang and Tamir Gonen and David S Eisenberg}, doi = {10.1074/jbc.M117.806109}, year = {2017}, date = {2017-02-23}, journal = {J. Biol. Chem.}, volume = {293}, number = {8}, pages = {2888--2902}, abstract = {Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aβ and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aβ(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aβ and hIAPP.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aβ and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aβ(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aβ and hIAPP. | |
de la Cruz, Jason M; Hattne, Johan ; Shi, Dan ; Rodriguez, Jose A; Reyes, Francis E; Gonen, Tamir Nature Protocol Exchange 2017. @online{2017_delacruz_b, title = {Micro- and nanocrystal preparation for MicroED and XFEL serial crystallography by fragmentation of imperfect crystals}, author = {de la Cruz, M. Jason and Hattne, Johan and Shi, Dan and Rodriguez, Jose A. and Reyes, Francis E. and Gonen, Tamir}, url = {https://cryoem.ucla.edu/wp-content/uploads/2017_delacruz_b.pdf}, doi = {10.1038/protex.2017.010}, year = {2017}, date = {2017-02-15}, journal = {Nature Protocol Exchange}, organization = {Nature Protocol Exchange}, abstract = {This protocol describes different techniques to fragment protein crystals for high-resolution structure determination by MicroED and serial crystallography at an X-ray free electron laser. These techniques involve agitation of the crystal of interest: 1) by vigorous pipetting, 2) by sonication, and 3) by vortexing. Aside from setup, the time needed to perform these steps ranges from seconds to minutes.}, keywords = {}, pubstate = {published}, tppubtype = {online} } This protocol describes different techniques to fragment protein crystals for high-resolution structure determination by MicroED and serial crystallography at an X-ray free electron laser. These techniques involve agitation of the crystal of interest: 1) by vigorous pipetting, 2) by sonication, and 3) by vortexing. Aside from setup, the time needed to perform these steps ranges from seconds to minutes. | |
de la Cruz, Jason M; Hattne, Johan; Shi, Dan; Seidler, Paul; Rodriguez, Jose; Reyes, Francis E; Sawaya, Michael R; Cascio, Duilio; Weiss, Simon C; Kim, Sun Kyung; Hinck, Cynthia S; Hinck, Andrew P; Calero, Guillermo; Eisenberg, David; Gonen, Tamir Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED Journal Article Nat. Methods, 14 (4), pp. 399–402, 2017. @article{pmid28192420, title = {Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED}, author = {Jason M de la Cruz and Johan Hattne and Dan Shi and Paul Seidler and Jose Rodriguez and Francis E Reyes and Michael R Sawaya and Duilio Cascio and Simon C Weiss and Sun Kyung Kim and Cynthia S Hinck and Andrew P Hinck and Guillermo Calero and David Eisenberg and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2017_delacruz_a.pdf, Main text}, doi = {10.1038/nmeth.4178}, year = {2017}, date = {2017-02-13}, journal = {Nat. Methods}, volume = {14}, number = {4}, pages = {399--402}, abstract = {Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography. | |
Krotee, Pascal; Rodriguez, Jose A; Sawaya, Michael R; Cascio, Duilio; Reyes, Francis E; Shi, Dan; Hattne, Johan; Nannenga, Brent L; Oskarsson, Marie E; Philipp, Stephan; Griner, Sarah; Jiang, Lin; Glabe, Charles G; Westermark, Gunilla T; Gonen, Tamir; Eisenberg, David S Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity Journal Article Elife, 6 , 2017. @article{pmid28045370, title = {Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity}, author = {Pascal Krotee and Jose A Rodriguez and Michael R Sawaya and Duilio Cascio and Francis E Reyes and Dan Shi and Johan Hattne and Brent L Nannenga and Marie E Oskarsson and Stephan Philipp and Sarah Griner and Lin Jiang and Charles G Glabe and Gunilla T Westermark and Tamir Gonen and David S Eisenberg}, url = {https://cryoem.ucla.edu/wp-content/uploads/2017_krotee.pdf, Main text}, doi = {10.7554/eLife.19273}, year = {2017}, date = {2017-01-03}, journal = {Elife}, volume = {6}, abstract = {hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP.}, keywords = {}, pubstate = {published}, tppubtype = {article} } hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP. | |
2016 |
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Nannenga, Brent L; Gonen, Tamir MicroED opens a new era for biological structure determination Journal Article Curr. Opin. Struct. Biol., 40 , pp. 128–135, 2016. @article{pmid27701014, title = {MicroED opens a new era for biological structure determination}, author = {Brent L Nannenga and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2016_nannengagonen.pdf, Main text}, doi = {10.1016/j.sbi.2016.09.007}, year = {2016}, date = {2016-10-01}, journal = {Curr. Opin. Struct. Biol.}, volume = {40}, pages = {128--135}, abstract = {In 2013 we unveiled the cryo-electron microscopy (CryoEM) method of MicroED, or three-dimensional (3D) electron diffraction of microscopic crystals. Here tiny 3D crystals of biological material are used in an electron microscope for diffraction data collection under cryogenic conditions. The data is indexed, integrated, merged and scaled using standard X-ray crystallography software to determine structures at atomic resolution. In this review we provide an overview of the MicroED method and compare it with other CryoEM methods.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In 2013 we unveiled the cryo-electron microscopy (CryoEM) method of MicroED, or three-dimensional (3D) electron diffraction of microscopic crystals. Here tiny 3D crystals of biological material are used in an electron microscope for diffraction data collection under cryogenic conditions. The data is indexed, integrated, merged and scaled using standard X-ray crystallography software to determine structures at atomic resolution. In this review we provide an overview of the MicroED method and compare it with other CryoEM methods. | |
Sawaya, Michael R; Rodriguez, Jose; Cascio, Duilio; Collazo, Michael J; Shi, Dan; Reyes, Francis E; Hattne, Johan; Gonen, Tamir; Eisenberg, David S Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED Journal Article Proc. Natl. Acad. Sci. U.S.A., 113 (40), pp. 11232–11236, 2016. @article{pmid27647903, title = {Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED}, author = {Michael R Sawaya and Jose Rodriguez and Duilio Cascio and Michael J Collazo and Dan Shi and Francis E Reyes and Johan Hattne and Tamir Gonen and David S Eisenberg}, url = {https://cryoem.ucla.edu/wp-content/uploads/2016_sawaya.pdf, Main text}, doi = {10.1073/pnas.1606287113}, year = {2016}, date = {2016-09-19}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {113}, number = {40}, pages = {11232--11236}, abstract = {Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods. | |
Bale, Jacob B; Gonen, Shane; Liu, Yuxi; Sheffler, William; Ellis, Daniel; Thomas, Chantz; Cascio, Duilio; Yeates, Todd O; Gonen, Tamir; King, Neil P; Baker, David Accurate design of megadalton-scale two-component icosahedral protein complexes Journal Article Science, 353 (6297), pp. 389–394, 2016. @article{pmid27463675, title = {Accurate design of megadalton-scale two-component icosahedral protein complexes}, author = {Jacob B Bale and Shane Gonen and Yuxi Liu and William Sheffler and Daniel Ellis and Chantz Thomas and Duilio Cascio and Todd O Yeates and Tamir Gonen and Neil P King and David Baker}, url = {https://cryoem.ucla.edu/wp-content/uploads/2016_bale.pdf, Main text}, doi = {10.1126/science.aaf8818}, year = {2016}, date = {2016-07-22}, journal = {Science}, volume = {353}, number = {6297}, pages = {389--394}, abstract = {Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines. | |
Hsia, Yang; Bale, Jacob B; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K; Nattermann, Una; Xu, Chunfu; Huang, Po‐Ssu.; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N; Gonen, Tamir; King, Neil P; Baker, David Design of a hyperstable 60-subunit protein icosahedron Journal Article Nature, 535 (7610), pp. 136–139, 2016. @article{pmid27309817, title = {Design of a hyperstable 60-subunit protein icosahedron}, author = {Yang Hsia and Jacob B Bale and Shane Gonen and Dan Shi and William Sheffler and Kimberly K Fong and Una Nattermann and Chunfu Xu and Po‐Ssu. Huang and Rashmi Ravichandran and Sue Yi and Trisha N Davis and Tamir Gonen and Neil P King and David Baker}, url = {https://cryoem.ucla.edu/wp-content/uploads/2016_hsia.pdf, Main text}, doi = {10.1038/nature18010}, year = {2016}, date = {2016-06-15}, journal = {Nature}, volume = {535}, number = {7610}, pages = {136--139}, abstract = {The dodecahedron [corrected] is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The dodecahedron [corrected] is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The dodecahedron [corrected] is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The dodecahedron [corrected] is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology. | |
Hattne, Johan; Shi, Dan; de la Cruz, Jason M; Reyes, Francis E; Gonen, Tamir Modeling truncated pixel values of faint reflections in MicroED images Journal Article J Appl Crystallogr, 49 (Pt 3), pp. 1029–1034, 2016. @article{pmid27275145, title = {Modeling truncated pixel values of faint reflections in MicroED images}, author = {Johan Hattne and Dan Shi and Jason M de la Cruz and Francis E Reyes and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/hattne_2016.pdf, Main text}, doi = {10.1107/S1600576716007196}, year = {2016}, date = {2016-06-01}, journal = {J Appl Crystallogr}, volume = {49}, number = {Pt 3}, pages = {1029--1034}, abstract = {The weak pixel counts surrounding the Bragg spots in a diffraction image are important for establishing a model of the background underneath the peak and estimating the reliability of the integrated intensities. Under certain circumstances, particularly with equipment not optimized for low-intensity measurements, these pixel values may be corrupted by corrections applied to the raw image. This can lead to truncation of low pixel counts, resulting in anomalies in the integrated Bragg intensities, such as systematically higher signal-to-noise ratios. A correction for this effect can be approximated by a three-parameter lognormal distribution fitted to the weakly positive-valued pixels at similar scattering angles. The procedure is validated by the improved refinement of an atomic model against structure factor amplitudes derived from corrected micro-electron diffraction (MicroED) images.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The weak pixel counts surrounding the Bragg spots in a diffraction image are important for establishing a model of the background underneath the peak and estimating the reliability of the integrated intensities. Under certain circumstances, particularly with equipment not optimized for low-intensity measurements, these pixel values may be corrupted by corrections applied to the raw image. This can lead to truncation of low pixel counts, resulting in anomalies in the integrated Bragg intensities, such as systematically higher signal-to-noise ratios. A correction for this effect can be approximated by a three-parameter lognormal distribution fitted to the weakly positive-valued pixels at similar scattering angles. The procedure is validated by the improved refinement of an atomic model against structure factor amplitudes derived from corrected micro-electron diffraction (MicroED) images. | |
Shi, Dan; Nannenga, Brent L; de la Cruz, Jason M; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir The collection of MicroED data for macromolecular crystallography Journal Article Nat Protoc, 11 (5), pp. 895–904, 2016. @article{pmid27077331, title = {The collection of MicroED data for macromolecular crystallography}, author = {Dan Shi and Brent L Nannenga and Jason M de la Cruz and Jinyang Liu and Steven Sawtelle and Guillermo Calero and Francis E Reyes and Johan Hattne and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2016_shi.pdf, Main text}, doi = {10.1038/nprot.2016.046}, year = {2016}, date = {2016-04-14}, journal = {Nat Protoc}, volume = {11}, number = {5}, pages = {895--904}, abstract = {The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals. | |
Meyer, Peter A; Socias, Stephanie; Key, Jason; Ransey, Elizabeth; Tjon, Emily C; Buschiazzo, Alejandro; Lei, Ming; Botka, Chris; Withrow, James; Neau, David; Rajashankar, Kanagalaghatta; Anderson, Karen S; Baxter, Richard H; Blacklow, Stephen C; Boggon, Titus J; Bonvin, Alexandre M J J; Borek, Dominika; Brett, Tom J; Caflisch, Amedeo; Chang, Chung-I; Chazin, Walter J; Corbett, Kevin D; Cosgrove, Michael S; Crosson, Sean; Dhe‐Paganon, Sirano; Cera, Enrico Di; Drennan, Catherine L; Eck, Michael J; Eichman, Brandt F; Fan, Qing R; Ferré‐D’Ámaré, Adrian R; Fromme, Christopher J; Garcia, Christopher K; Gaudet, Rachelle; Gong, Peng; Harrison, Stephen C; Heldwein, Ekaterina E; Jia, Zongchao; Keenan, Robert J; Kruse, Andrew C; Kvansakul, Marc; McLellan, Jason S; Modis, Yorgo; Nam, Yunsun; Otwinowski, Zbyszek; Pai, Emil F; Pereira, Pedro José Barbosa; Petosa, Carlo; Raman, C S; Rapoport, Tom A; Roll‐Mecak, Antonina; Rosen, Michael K; Rudenko, Gabby; Schlessinger, Joseph; Schwartz, Thomas U; Shamoo, Yousif; Sondermann, Holger; Tao, Yizhi J; Tolia, Niraj H; Tsodikov, Oleg V; Westover, Kenneth D; Wu, Hao; Foster, Ian; Fraser, James S; Maia, Felipe R N C; Gonen, Tamir; Kirchhausen, Tom; Diederichs, Kay; Crosas, Mercè; Sliz, Piotr Data publication with the structural biology data grid supports live analysis Journal Article Nat Commun, 7 , pp. 10882, 2016. @article{pmid26947396, title = {Data publication with the structural biology data grid supports live analysis}, author = {Peter A Meyer and Stephanie Socias and Jason Key and Elizabeth Ransey and Emily C Tjon and Alejandro Buschiazzo and Ming Lei and Chris Botka and James Withrow and David Neau and Kanagalaghatta Rajashankar and Karen S Anderson and Richard H Baxter and Stephen C Blacklow and Titus J Boggon and Alexandre M J J Bonvin and Dominika Borek and Tom J Brett and Amedeo Caflisch and Chung-I Chang and Walter J Chazin and Kevin D Corbett and Michael S Cosgrove and Sean Crosson and Sirano Dhe‐Paganon and Enrico Di Cera and Catherine L Drennan and Michael J Eck and Brandt F Eichman and Qing R Fan and Adrian R Ferré‐D’Ámaré and Christopher J Fromme and Christopher K Garcia and Rachelle Gaudet and Peng Gong and Stephen C Harrison and Ekaterina E Heldwein and Zongchao Jia and Robert J Keenan and Andrew C Kruse and Marc Kvansakul and Jason S McLellan and Yorgo Modis and Yunsun Nam and Zbyszek Otwinowski and Emil F Pai and Pedro José Barbosa Pereira and Carlo Petosa and C S Raman and Tom A Rapoport and Antonina Roll‐Mecak and Michael K Rosen and Gabby Rudenko and Joseph Schlessinger and Thomas U Schwartz and Yousif Shamoo and Holger Sondermann and Yizhi J Tao and Niraj H Tolia and Oleg V Tsodikov and Kenneth D Westover and Hao Wu and Ian Foster and James S Fraser and Felipe R N C Maia and Tamir Gonen and Tom Kirchhausen and Kay Diederichs and Mercè Crosas and Piotr Sliz}, url = {https://cryoem.ucla.edu/wp-content/uploads/2016_meyer.pdf, Main text}, doi = {10.1038/ncomms10882}, year = {2016}, date = {2016-03-07}, journal = {Nat Commun}, volume = {7}, pages = {10882}, abstract = {Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis. | |
Rodriguez, J A; Gonen, T High-Resolution Macromolecular Structure Determination by MicroED, a cryo-EM Method Book Chapter Methods in Enzymology, 579 , Chapter 14, pp. 369–392, 2016. @inbook{pmid27572734, title = {High-Resolution Macromolecular Structure Determination by MicroED, a cryo-EM Method}, author = {J A Rodriguez and T Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2016_rodriguezgonen.pdf, Main text}, doi = {10.1016/bs.mie.2016.04.017}, year = {2016}, date = {2016-01-01}, booktitle = {Methods in Enzymology}, issuetitle = {579}, journal = {Meth. Enzymol.}, volume = {579}, pages = {369--392}, chapter = {14}, abstract = {Microelectron diffraction (MicroED) is a new cryo-electron microscopy (cryo-EM) method capable of determining macromolecular structures at atomic resolution from vanishingly small 3D crystals. MicroED promises to solve atomic resolution structures from even the tiniest of crystals, less than a few hundred nanometers thick. MicroED complements frontier advances in crystallography and represents part of the rebirth of cryo-EM that is making macromolecular structure determination more accessible for all. Here we review the concept and practice of MicroED, for both the electron microscopist and crystallographer. Where other reviews have addressed specific details of the technique (Hattne et al., 2015; Shi et al., 2016; Shi, Nannenga, Iadanza, & Gonen, 2013), we aim to provide context and highlight important features that should be considered when performing a MicroED experiment.}, keywords = {}, pubstate = {published}, tppubtype = {inbook} } Microelectron diffraction (MicroED) is a new cryo-electron microscopy (cryo-EM) method capable of determining macromolecular structures at atomic resolution from vanishingly small 3D crystals. MicroED promises to solve atomic resolution structures from even the tiniest of crystals, less than a few hundred nanometers thick. MicroED complements frontier advances in crystallography and represents part of the rebirth of cryo-EM that is making macromolecular structure determination more accessible for all. Here we review the concept and practice of MicroED, for both the electron microscopist and crystallographer. Where other reviews have addressed specific details of the technique (Hattne et al., 2015; Shi et al., 2016; Shi, Nannenga, Iadanza, & Gonen, 2013), we aim to provide context and highlight important features that should be considered when performing a MicroED experiment. | |
2015 |
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Rodriguez, Jose A; Ivanova, Magdalena I; Sawaya, Michael R; Cascio, Duilio; Reyes, Francis E; Shi, Dan; Sangwan, Smriti; Guenther, Elizabeth L; Johnson, Lisa M; Zhang, Meng; Jiang, Lin; Arbing, Mark A; Nannenga, Brent L; Hattne, Johan; Whitelegge, Julian; Brewster, Aaron S; Messerschmidt, Marc; Boutet, Sébastien; Sauter, Nicholas K; Gonen, Tamir; Eisenberg, David S Structure of the toxic core of α-synuclein from invisible crystals Journal Article Nature, 525 (7570), pp. 486–490, 2015. @article{pmid26352473, title = {Structure of the toxic core of α-synuclein from invisible crystals}, author = {Jose A Rodriguez and Magdalena I Ivanova and Michael R Sawaya and Duilio Cascio and Francis E Reyes and Dan Shi and Smriti Sangwan and Elizabeth L Guenther and Lisa M Johnson and Meng Zhang and Lin Jiang and Mark A Arbing and Brent L Nannenga and Johan Hattne and Julian Whitelegge and Aaron S Brewster and Marc Messerschmidt and Sébastien Boutet and Nicholas K Sauter and Tamir Gonen and David S Eisenberg}, url = {https://cryoem.ucla.edu/wp-content/uploads/2015rodriguez.pdf, Main text}, doi = {10.1038/nature15368}, year = {2015}, date = {2015-09-09}, journal = {Nature}, volume = {525}, number = {7570}, pages = {486--490}, abstract = {The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils. | |
Bale, Jacob B; Park, Rachel U; Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Cascio, Duilio; King, Neil P; Yeates, Todd O; Baker, David Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression Journal Article Protein Sci., 24 (10), pp. 1695–1701, 2015. @article{pmid26174163, title = {Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression}, author = {Jacob B Bale and Rachel U Park and Yuxi Liu and Shane Gonen and Tamir Gonen and Duilio Cascio and Neil P King and Todd O Yeates and David Baker}, url = {https://cryoem.ucla.edu/wp-content/uploads/2015_bale.pdf, Main text}, doi = {10.1002/pro.2748}, year = {2015}, date = {2015-07-15}, journal = {Protein Sci.}, volume = {24}, number = {10}, pages = {1695--1701}, abstract = {We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials. | |
Hattne, Johan; Reyes, Francis E; Nannenga, Brent L; Shi, Dan; de la Cruz, Jason M; Leslie, Andrew G W; Gonen, Tamir MicroED data collection and processing Journal Article Acta Crystallogr A Found Adv, 71 (Pt 4), pp. 353–360, 2015. @article{pmid26131894, title = {MicroED data collection and processing}, author = {Johan Hattne and Francis E Reyes and Brent L Nannenga and Dan Shi and Jason M de la Cruz and Andrew G W Leslie and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2015_hattne.pdf, Main text}, doi = {10.1107/S2053273315010669}, year = {2015}, date = {2015-07-01}, journal = {Acta Crystallogr A Found Adv}, volume = {71}, number = {Pt 4}, pages = {353--360}, abstract = {MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.}, keywords = {}, pubstate = {published}, tppubtype = {article} } MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges. | |