2019
Hattne, Johan; Martynowycz, Michael W; Penczek, Pawel A; Gonen, Tamir
MicroED with the Falcon III direct electron detector
In: IUCrJ, vol. 6, no. 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; Jing, Liang; Shi, Dan; Gonen, Tamir; Liu, Wei; Nannenga, Brent L.
Structure determination from lipidic cubic phase embedded microcrystals by MicroED
bioRxiv, 2019.
@unpublished{Zhu2019,
title = {Structure determination from lipidic cubic phase embedded microcrystals by MicroED},
author = {Lan Zhu and Guanhong Bu and Liang Jing and Dan Shi and Tamir Gonen and Wei Liu and Brent L. Nannenga},
url = {http://biorxiv.org/lookup/doi/10.1101/724575},
doi = {10.1101/724575},
year = {2019},
date = {2019-08-04},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
In: Nat Commun, vol. 10, no. 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
In: Science, vol. 365, no. 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
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
In: Ultramicroscopy, vol. 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)
In: Nat. Methods, vol. 16, no. 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.
Hattne, Johan; Martynowycz, Michael W.; Gonen, Tamir
MicroED with the Falcon III direct electron detector
bioRxiv, 2019.
@unpublished{Hattne2019,
title = {MicroED with the Falcon III direct electron detector},
author = {Johan Hattne and Michael W. Martynowycz and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/615484},
doi = {10.1101/615484},
year = {2019},
date = {2019-04-23},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract Microcrystal electron diffraction (MicroED) combines crystallography and electron cryomicroscopy (cryo-EM) into a method that can be used for high-resolution structure determination. In MicroED nanosized crystals, often intractable by other techniques, are probed by high-energy electrons in a transmission electron microscope and the diffracted signal is recorded on an electron detector. Since only a small number of different detectors have been used for MicroED measurements in the past, their impact on data quality has not been investigated. Here we evaluate two different cameras using crystals of the well-characterized serine protease proteinase K. Compared to previously used equipment, the Falcon III direct electron detector and the CMOS-based CetaD camera can collect complete datasets both faster and using lower total exposure. As an effect of the lower dose, radiation damage is reduced, which is confirmed in both real and reciprocal space. The increased speed and lower exposure requirements have implications on model quality and the prospects for further automation of MicroED. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Martynowycz, Michael W.; Zhao, Wei; Hattne, Johan; Jensen, Grant J.; Gonen, Tamir
Qualitative analyses of polishing and pre-coating FIB milled crystals for MicroED
bioRxiv, 2019.
@unpublished{Martynowycz2019b,
title = {Qualitative analyses of polishing and pre-coating FIB milled crystals for MicroED},
author = {Michael W. Martynowycz and Wei Zhao and Johan Hattne and Grant J. Jensen and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/613042},
doi = {10.1101/613042},
year = {2019},
date = {2019-04-18},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Summary 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) can thin even very large 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 platinum pre-coated and uncoated samples. Our results suggest that pre-coating 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/noise ratio, higher resolution and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
In: Structure, vol. 27, no. 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
In: IUCrJ, vol. 6, no. 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 Å by microfocus X-ray crystallography and to 0.9 Å 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 Å by microfocus X-ray crystallography and to 0.9 Å 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
In: Proc. Natl. Acad. Sci. U.S.A., vol. 116, no. 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
In: Elife, vol. 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
Purdy, Michael D; Shi, Dan; Chrustowicz, Jakub; Hattne, Johan; Gonen, Tamir; Yeager, Mark
In: Proc. Natl. Acad. Sci. U.S.A., vol. 115, no. 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.
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 from the ice nucleation protein InaZ
bioRxiv, 2018.
@unpublished{Zee2018,
title = {Homochiral and racemic MicroED structures of a peptide 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},
url = {http://biorxiv.org/lookup/doi/10.1101/473116},
doi = {10.1101/473116},
year = {2018},
date = {2018-11-19},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract The ice nucleation protein InaZ of Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. We determine ab initio structures of this repeat segment, resolved to 1.1Å by microfocus x-ray crystallography and 0.9Å by the cryoEM method MicroED, from both racemic and homochiral crystals. We evaluate the benefits of racemic protein crystals for structure determination by MicroED and confirm that phase restriction introduced by crystal centrosymmetry increases the number of successful trials during ab initio phasing of electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel beta 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. Our determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with sub-micron sized crystals. Synopsis The atomic asymmetry, left or right handedness, present in macromolecules and first described by Pasteur in his experiments with tartaric acid, is evident even in complex molecular assemblies like amyloid fibrils. Here, using the cryoEM method MicroED, we show that a segment from the ice nucleation protein InaZ assembles into homochiral and racemic water-binding amyloid protofibrils.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
In: ACS Cent Sci, vol. 4, no. 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 Å) 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 Å) crystal structures in minutes.
de la Cruz, M. Jason; Martynowycz, Michael W.; Hattne, Johan; Gonen, Tamir
MicroED data collection with SerialEM
bioRxiv, 2018.
@unpublished{JasondelaCruz2018,
title = {MicroED data collection with SerialEM},
author = {M. Jason de la Cruz and Michael W. Martynowycz and Johan Hattne and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/446658},
doi = {10.1101/446658},
year = {2018},
date = {2018-10-22},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract We developed a procedure for the cryoEM method MicroED using SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. More than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
bioRxiv, 2018.
@unpublished{Martynowycz2018,
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},
url = {http://biorxiv.org/lookup/doi/10.1101/425611},
doi = {10.1101/425611},
year = {2018},
date = {2018-09-24},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Jinming, Ma; Lei, Hsiang-Ting; Gonen, Tamir
A conformational change in the N terminus of SLC38A9 signals mTORC1 activation
bioRxiv, 2018.
@unpublished{Jinming2018,
title = {A conformational change in the N terminus of SLC38A9 signals mTORC1 activation},
author = {Ma Jinming and Hsiang-Ting Lei and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/339937},
doi = {10.1101/339937},
year = {2018},
date = {2018-06-06},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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. One Sentence Summary N-plug inserted state of SLC38A9 reveals mechanisms of mTORC1 activation and arginine-enhanced luminal amino acids efflux.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Lei, Hsiang-Ting; Ma, Jinming; Martinez, Silvia Sanchez; Gonen, Tamir
Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state
In: Nat. Struct. Mol. Biol., vol. 25, no. 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
In: Commun Biol, vol. 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
In: Structure, vol. 26, no. 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
bioRxiv, 2018.
@unpublished{Ma2018,
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 Sarhan and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/293811},
doi = {10.1101/293811},
year = {2018},
date = {2018-04-03},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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 fromMethanococcus 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. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Yeates, Todd O
Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system
In: Proc. Natl. Acad. Sci. U.S.A., vol. 115, no. 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.
Lei, Hsiang-Ting; Ma, Jinming; Martinez, Silvia Sanchez; Gonen, Tamir
Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state
bioRxiv, 2018.
@unpublished{Lei2018,
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},
url = {http://biorxiv.org/lookup/doi/10.1101/288233},
doi = {10.1101/288233},
year = {2018},
date = {2018-03-24},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Amino acid-dependent activation of mechanistic target of rapamycin complex 1 (mTORC1) is essential to reflect nutrient availabilities in cells for cell growth and metabolism1 . Solute carrier 38 family A member 9 (SLC38A9) is the lysosomal transporter responsible for amino acid sensing in the mTORC1 signaling pathway2–4 . 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 the transmembrane helix 1 (TM1) of SLC38A9 which was anchored at the grove between transmembrane helix 5 and 7 inside the transporter. The key motif WNTMM on TM1, contributing to the anchoring interactions, is highly conserved in various species. Mutations in WNTMM motif abolished arginine transport by SLC38A9. The underlying mechanism of substrate binding is critical for both sensitizing mTORC1 signaling pathway to amino acids and for maintaining amino acid homeostasis across lysosomal membranes2 . },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
In: Nat. Struct. Mol. Biol., vol. 25, no. 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
In: Curr Opin Colloid Interface Sci, vol. 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
In: Science, vol. 359, no. 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
In: Emerg Top Life Sci, vol. 2, no. 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.
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
bioRxiv, 2018.
@unpublished{Hattne2018,
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},
url = {http://biorxiv.org/lookup/doi/10.1101/258178},
doi = {10.1101/258178},
year = {2018},
date = {2018-02-01},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Summary Micro-crystal electron diffraction (MicroED) is an emerging method in cryo-EM for structure determination using nanocrystals. 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. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
In: Nat. Struct. Mol. Biol., vol. 25, no. 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-ångström-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-ångström-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
Purdy, Michael D.; Shi, Dan; Chrustowicz, Jakub; Hattne, Johan; Gonen, Tamir; Yeager, Mark
bioRxiv, 2017.
@unpublished{Purdy2017,
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},
url = {http://biorxiv.org/lookup/doi/10.1101/241182},
doi = {10.1101/241182},
year = {2017},
date = {2017-12-30},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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, three-dimensional 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 6-helix bundle via both electrostatic interactions with the dimethysuccinyl 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. One-sentence summary The MicroED structure of HIV-1 Gag CTD-SP1 at 2.9 Å resolution reveals the molecular basis for binding of the maturation inhibitor bevirimat that will inform further drug discovery efforts.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
In: Nat Chem, vol. 10, no. 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.
Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Yeates, Todd O.
Near-Atomic Cryo-EM Imaging of a Small Protein Displayed on a Designed Scaffolding System
bioRxiv, 2017.
@unpublished{Liu2017,
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},
url = {http://biorxiv.org/lookup/doi/10.1101/212233},
doi = {10.1101/212233},
year = {2017},
date = {2017-11-01},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract Current single particle electron cryo-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 amenable to 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 cryoEM to proteins of typical size in the cell. Significance statement New electron microscopy methods are making it possible to view the structures of large proteins and nucleic acid complexes at atomic detail, but the methods are difficult to apply to molecules smaller than about 50 kDa, which is larger than the size of the average protein in the cell. The present work demonstrates that a protein much smaller than that limit can be successfully visualized when it is attached to a large protein scaffold designed to hold 12 copies of the attached protein in symmetric and rigidly defined orientations. The small protein chosen for attachment and visualization can be modified to bind to other diverse proteins, opening up a new avenue for imaging cellular proteins by cryo-EM.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
In: J Phys Chem Lett, vol. 8, no. 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
In: Curr. Opin. Struct. Biol., vol. 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
In: J. Am. Chem. Soc., vol. 139, no. 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
In: Protein Sci., vol. 26, no. 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.; Goldschmidt, Lukasz; Rodriguez, Jose A.; Cascio, Duilio; Gonen, Tamir; Eisenberg, David S.
Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets
bioRxiv, 2017.
@unpublished{Hughes2017,
title = {Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets},
author = {Michael P. Hughes and Michael R. Sawaya and Lukasz Goldschmidt and Jose A. Rodriguez and Duilio Cascio and Tamir Gonen and David S. Eisenberg},
url = {http://biorxiv.org/lookup/doi/10.1101/153817},
doi = {10.1101/153817},
year = {2017},
date = {2017-06-22},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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. One Sentence Summary Atomic structures show transient membraneless organelles of cells formed by a new type of protein interaction akin to pathogenic amyloid fibrils.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
de la Cruz, M. Jason; 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; de la Cruz, M. Jason; Hattne, Johan; Shi, Dan; Cascio, Duilio; Rodriguez, Jose; Gonen, Tamir
MicroED Structures from Micrometer Thick Protein Crystals
bioRxiv, 2017.
@unpublished{Martynowycz2017,
title = {MicroED Structures from Micrometer Thick Protein Crystals},
author = {Michael W. Martynowycz and Calina Glynn and Jennifer Miao and M. Jason de la Cruz and Johan Hattne and Dan Shi and Duilio Cascio and Jose Rodriguez and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/152504},
doi = {10.1101/152504},
year = {2017},
date = {2017-06-20},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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
In: J. Biol. Chem., vol. 293, no. 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, M. Jason; 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
In: Nat. Methods, vol. 14, no. 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
In: Elife, vol. 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.
Gonen, Tamir (Ed.)
Challenges in Structural Biology
HHMI Janelia Research Campus, 2017.
@conference{2017Janelia,
title = {Challenges in Structural Biology},
editor = {Tamir Gonen},
year = {2017},
date = {2017-00-00},
publisher = {Janelia Research Campus},
organization = {HHMI},
keywords = {},
pubstate = {published},
tppubtype = {conference}
}
2016
Nannenga, Brent L; Gonen, Tamir
MicroED opens a new era for biological structure determination
In: Curr. Opin. Struct. Biol., vol. 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
In: Proc. Natl. Acad. Sci. U.S.A., vol. 113, no. 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
In: Science, vol. 353, no. 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
In: Nature, vol. 535, no. 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.