Investigator, Howard Hughes Medical Institute at UCLA
Tamir Gonen is a membrane biophysicist and an expert in crystallography and cryo-EM. Gonen is a professor of Biological Chemistry and Physiology at the David Geffen School of Medicine of the University of California, Los Angeles, an Investigator of the Howard Hughes Medical Institute, and a Member of the Royal Society of New Zealand. He received a Career Development Award from the American Diabetes Association and was an Early Career Scientist of HHMI. Gonen served on several study sections of the National Institutes of Health and acted as ad hoc reviewer for several international funding agencies. In 2011 while leading a lab at the HHMI Janelia Research Campus he began developing microcrystal electron diffraction (MicroED) as a new method for structural biology. In 2017 Dr Gonen moved his laboratory to the David Geffen School of Medicine of the University of California, Los Angeles as an Investigator of the Howard Hughes Medical Institute and a Professor of Biological Chemistry and Physiology, where he continues studying membrane protein structure and function using cryo-EM and MicroED. With this method Dr Gonen has pushed the boundaries of cryo-EM and determined several previously unknown structures at resolutions better than 1 Å. Gonen authored more than 120 publications and several of his past trainees are now faculty around the world at top universities.
Honors
| 1996 | Dean’s list—Organic Chemistry, University of Auckland, New Zealand |
| 1996 | Dean’s list—Inorganic Chemistry, University of Auckland, New Zealand |
| 1997 | Center for Gene Technology Research Scholarship, University of Auckland, New Zealand |
| 1997 | Dean’s List—Inorganic Chemistry, University of Auckland, New Zealand |
| 1998 | Senior prize in Biological Sciences, University of Auckland, New Zealand |
| 1998 | First class honors in Biological Sciences, University of Auckland, New Zealand |
| 1999 | University of Auckland Doctoral Scholarship, University of Auckland, New Zealand |
| 2000 | Contestable Travel Fund Award, University of Auckland, New Zealand |
| 2001 | Contestable Travel Fund Award, University of Auckland, New Zealand |
| 2009 | American Diabetes Association Career Development Award |
| 2009 | Howard Hughes Medical Institute Early Career Scientist |
| 2010 | New investigator, Science in Medicine Lecture |
| 2012 | Member, The Royal Society of New Zealand |
| 2017 | Investigator, Howard Hughes Medical Institute |
| 2018 | Chair elect, Biophysical Society cryo-EM subgroup |
Publications
2021
Martynowycz, Michael W; Clabbers, Max T B; Unge, Johan; Hattne, Johan; Gonen, Tamir
Benchmarking the ideal sample thickness in cryo-EM Journal Article
In: Proc Natl Acad Sci U S A, 118 (49), 2021, ISSN: 1091-6490.
@article{pmid34873060,
title = {Benchmarking the ideal sample thickness in cryo-EM},
author = {Michael W Martynowycz and Max T B Clabbers and Johan Unge and Johan Hattne and Tamir Gonen},
doi = {10.1073/pnas.2108884118},
issn = {1091-6490},
year = {2021},
date = {2021-12-01},
urldate = {2021-12-01},
journal = {Proc Natl Acad Sci U S A},
volume = {118},
number = {49},
abstract = {The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.},
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The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.
Clark, Lisa J.; Bu, Guanhong; Nannenga, Brent L.; Gonen, Tamir
MicroED for the study of protein–ligand interactions and the potential for drug discovery Journal Article
In: Nat Rev Chem, 5 , pp. 853–858, 2021.
@article{Clark2021,
title = {MicroED for the study of protein–ligand interactions and the potential for drug discovery},
author = {Lisa J. Clark and Guanhong Bu and Brent L. Nannenga and Tamir Gonen},
doi = {10.1038/s41570-021-00332-y},
year = {2021},
date = {2021-10-27},
urldate = {2021-10-27},
journal = {Nat Rev Chem},
volume = {5},
pages = {853–858},
abstract = {Microcrystal electron diffraction (MicroED) is an electron cryo-microscopy (cryo-EM) technique used to determine molecular structures with crystals that are a millionth the size needed for traditional single-crystal X-ray crystallography. An exciting use of MicroED is in drug discovery and development, where it can be applied to the study of proteins and small molecule interactions, and for structure determination of natural products. The structures are then used for rational drug design and optimization. In this Perspective, we discuss the current applications of MicroED for structure determination of protein–ligand complexes and potential future applications in drug discovery.},
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Microcrystal electron diffraction (MicroED) is an electron cryo-microscopy (cryo-EM) technique used to determine molecular structures with crystals that are a millionth the size needed for traditional single-crystal X-ray crystallography. An exciting use of MicroED is in drug discovery and development, where it can be applied to the study of proteins and small molecule interactions, and for structure determination of natural products. The structures are then used for rational drug design and optimization. In this Perspective, we discuss the current applications of MicroED for structure determination of protein–ligand complexes and potential future applications in drug discovery.
Martynowycz, Michael W; Gonen, Tamir
Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED) Journal Article
In: STAR Protoc, 2 (3), pp. 100686, 2021, ISSN: 2666-1667.
@article{pmid34382014,
title = {Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED)},
author = {Michael W Martynowycz and Tamir Gonen},
doi = {10.1016/j.xpro.2021.100686},
issn = {2666-1667},
year = {2021},
date = {2021-09-17},
journal = {STAR Protoc},
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abstract = {We present an in-depth protocol to reproducibly prepare crystalline lamellae from protein crystals for subsequent microcrystal electron diffraction (MicroED) experiments. This protocol covers typical soluble proteins and membrane proteins embedded in dense media. Following these steps will allow the user to prepare crystalline lamellae for protein structure determination by MicroED. For complete details on the use and execution of this protocol, please refer to Martynowycz et al. (2019a, 2020a).},
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We present an in-depth protocol to reproducibly prepare crystalline lamellae from protein crystals for subsequent microcrystal electron diffraction (MicroED) experiments. This protocol covers typical soluble proteins and membrane proteins embedded in dense media. Following these steps will allow the user to prepare crystalline lamellae for protein structure determination by MicroED. For complete details on the use and execution of this protocol, please refer to Martynowycz et al. (2019a, 2020a).
Martynowycz, Michael W.; Shiriaeva, Anna; Ge, Xuanrui; Hattne, Johan; Nannenga, Brent L.; Cherezov, Vadim; Gonen, Tamir
MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP Journal Article
In: Proc Natl Acad Sci U S A, 118 (36), pp. e2106041118, 2021.
@article{2020_Martynowycz,
title = {MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP},
author = {Martynowycz, Michael W. and Shiriaeva, Anna and Ge, Xuanrui and Hattne, Johan and Nannenga, Brent L. and Cherezov, Vadim and Gonen, Tamir},
doi = {10.1073/pnas.2106041118},
year = {2021},
date = {2021-09-07},
urldate = {2021-09-07},
journal = {Proc Natl Acad Sci U S A},
volume = {118},
number = {36},
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organization = {bioRxiv},
abstract = {G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A2A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods.},
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G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A2A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods.
Mu, Xuelang; Gillman, Cody; Nguyen, Chi; Gonen, Tamir
An Overview of Microcrystal Electron Diffraction (MicroED) Journal Article
In: Annu Rev Biochem, 90 , pp. 431–450, 2021, ISSN: 1545-4509.
@article{pmid34153215,
title = {An Overview of Microcrystal Electron Diffraction (MicroED)},
author = {Xuelang Mu and Cody Gillman and Chi Nguyen and Tamir Gonen},
doi = {10.1146/annurev-biochem-081720-020121},
issn = {1545-4509},
year = {2021},
date = {2021-06-20},
urldate = {2021-06-00},
journal = {Annu Rev Biochem},
volume = {90},
pages = {431--450},
abstract = {The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.},
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The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.
Lei, Hsiang-Ting; Mu, Xuelang; Hattne, Johan; Gonen, Tamir
A conformational change in the N Terminus of SLC38A9 signals mTORC1 activation Journal Article
In: Structure, 29 (5), pp. 426–432.e8, 2021.
@article{Lei2020,
title = {A conformational change in the N Terminus of SLC38A9 signals mTORC1 activation},
author = {Hsiang-Ting Lei and Xuelang Mu and Johan Hattne and Tamir Gonen},
doi = {10.1016/j.str.2020.11.014},
year = {2021},
date = {2021-05-06},
urldate = {2021-05-06},
journal = {Structure},
volume = {29},
number = {5},
pages = {426--432.e8},
abstract = {mTORC1 is a central hub that integrates environmental cues, such as cellular stresses and nutrient availability to modulate metabolism and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, emerged as a sensor for luminal arginine and as an activator of mTORC1. The amino acid-mediated activation of mTORC1 is regulated by the N-terminal domain of SLC38A9. Here, we determined the crystal structure of zebrafish SLC38A9 (drSLC38A9) and found the N-terminal fragment inserted deep within the transporter, bound in the substrate-binding pocket where normally arginine would bind. This represents a significant conformational change of the N-terminal domain (N-plug) when compared with our recent arginine-bound structure of drSLC38A9. We propose a ball-and-chain model for mTORC1 activation, where N-plug insertion and Rag GTPase binding with SLC38A9 is regulated by luminal arginine levels. This work provides important insights into nutrient sensing by SLC38A9 to activate the mTORC1 pathways in response to dietary amino acids.},
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mTORC1 is a central hub that integrates environmental cues, such as cellular stresses and nutrient availability to modulate metabolism and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, emerged as a sensor for luminal arginine and as an activator of mTORC1. The amino acid-mediated activation of mTORC1 is regulated by the N-terminal domain of SLC38A9. Here, we determined the crystal structure of zebrafish SLC38A9 (drSLC38A9) and found the N-terminal fragment inserted deep within the transporter, bound in the substrate-binding pocket where normally arginine would bind. This represents a significant conformational change of the N-terminal domain (N-plug) when compared with our recent arginine-bound structure of drSLC38A9. We propose a ball-and-chain model for mTORC1 activation, where N-plug insertion and Rag GTPase binding with SLC38A9 is regulated by luminal arginine levels. This work provides important insights into nutrient sensing by SLC38A9 to activate the mTORC1 pathways in response to dietary amino acids.
Danelius, E; Gonen, T
Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED Book Chapter
In: Owens, Raymond J. (Ed.): 2305 , pp. 323–342, 2021.
@inbook{pmid33950397,
title = {Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED},
author = {E Danelius and T Gonen},
editor = {Raymond J. Owens},
doi = {10.1007/978-1-0716-1406-8_16},
year = {2021},
date = {2021-05-06},
journal = {Methods Mol Biol},
volume = {2305},
pages = {323--342},
series = {Methods in Molecular Biology},
abstract = {Microcrystal Electron Diffraction (MicroED) is the newest cryo-electron microscopy (cryo-EM) method, with over 70 protein, peptide, and several small organic molecule structures already determined. In MicroED, micro- or nanocrystalline samples in solution are deposited on electron microscopy grids and examined in a cryo-electron microscope, ideally under cryogenic conditions. Continuous rotation diffraction data are collected and then processed using conventional X-ray crystallography programs. The protocol outlined here details how to obtain and identify the nanocrystals, how to set up the microscope for screening and for MicroED data collection, and how to collect and process data to complete high-resolution structures. For well-behaving crystals with high-resolution diffraction in cryo-EM, the entire process can be achieved in less than an hour.},
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Microcrystal Electron Diffraction (MicroED) is the newest cryo-electron microscopy (cryo-EM) method, with over 70 protein, peptide, and several small organic molecule structures already determined. In MicroED, micro- or nanocrystalline samples in solution are deposited on electron microscopy grids and examined in a cryo-electron microscope, ideally under cryogenic conditions. Continuous rotation diffraction data are collected and then processed using conventional X-ray crystallography programs. The protocol outlined here details how to obtain and identify the nanocrystals, how to set up the microscope for screening and for MicroED data collection, and how to collect and process data to complete high-resolution structures. For well-behaving crystals with high-resolution diffraction in cryo-EM, the entire process can be achieved in less than an hour.
Martynowycz, M W; Gonen, T
Studying Membrane Protein Structures by MicroED Book Chapter
In: Schmidt-Krey, Ingeborg; Gumbart, James C. (Ed.): 2302 , pp. 137–151, 2021.
@inbook{pmid33877626,
title = {Studying Membrane Protein Structures by MicroED},
author = {M W Martynowycz and T Gonen},
editor = {Ingeborg Schmidt-Krey and James C. Gumbart},
doi = {10.1007/978-1-0716-1394-8_8},
year = {2021},
date = {2021-04-21},
journal = {Methods Mol Biol},
volume = {2302},
pages = {137--151},
series = {Methods in Molecular Biology},
abstract = {Microcrystal electron diffraction (MicroED) enables atomic resolution structures to be determined from vanishingly small crystals. Soluble proteins typically grow crystals that are tens to hundreds of microns in size for X-ray crystallography. But membrane protein crystals often grow crystals that are too small for X-ray diffraction and yet too large for MicroED. These crystals are often formed in thick, viscous media that challenge traditional cryoEM grid preparation. Here, we describe two approaches for preparing membrane protein crystals for MicroED data collection: application of a crystal slurry directly to EM grids, and focused ion beam milling in a Scanning Electron Microscope (FIB-SEM). We summarize the case of preparing an ion channel, NaK, and the workflow of focused ion-beam milling. By milling away the excess media and crystalline material, crystals of any size may be prepared for MicroED. Finally, an energy filter may be used to help minimize inelastic scattering leading to lower noise on recorded images.},
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Microcrystal electron diffraction (MicroED) enables atomic resolution structures to be determined from vanishingly small crystals. Soluble proteins typically grow crystals that are tens to hundreds of microns in size for X-ray crystallography. But membrane protein crystals often grow crystals that are too small for X-ray diffraction and yet too large for MicroED. These crystals are often formed in thick, viscous media that challenge traditional cryoEM grid preparation. Here, we describe two approaches for preparing membrane protein crystals for MicroED data collection: application of a crystal slurry directly to EM grids, and focused ion beam milling in a Scanning Electron Microscope (FIB-SEM). We summarize the case of preparing an ion channel, NaK, and the workflow of focused ion-beam milling. By milling away the excess media and crystalline material, crystals of any size may be prepared for MicroED. Finally, an energy filter may be used to help minimize inelastic scattering leading to lower noise on recorded images.
Martynowycz, M W; Gonen, T
Microcrystal Electron Diffraction of Small Molecules Journal Article
In: J Vis Exp, (169), 2021.
@article{pmid33779618,
title = {Microcrystal Electron Diffraction of Small Molecules},
author = {M W Martynowycz and T Gonen},
doi = {10.3791/62313},
year = {2021},
date = {2021-03-15},
journal = {J Vis Exp},
number = {169},
abstract = {A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.},
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A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.
Danelius, E; Halaby, S; van der Donk, W A; Gonen, T
MicroED in natural product and small molecule research Journal Article
In: Nat Prod Rep, 38 (3), 2021.
@article{pmid32939523,
title = {MicroED in natural product and small molecule research},
author = {E Danelius and S Halaby and W A van der Donk and T Gonen},
doi = {10.1039/d0np00035c},
year = {2021},
date = {2021-03-01},
urldate = {2020-09-01},
journal = {Nat Prod Rep},
volume = {38},
number = {3},
abstract = {The electron cryo-microscopy (cryo-EM) method Microcrystal Electron Diffraction (MicroED) allows the collection of high-resolution structural data from vanishingly small crystals that appear like amorphous powders or very fine needles. Since its debut in 2013, data collection and analysis schemes have been fine-tuned, and there are currently close to 100 structures determined by MicroED. Although originally developed to study proteins, MicroED is also very powerful for smaller systems, with some recent and very promising examples from the field of natural products. Herein, we review what has been achieved so far and provide examples of natural product structures, as well as demonstrate the expected future impact of MicroED to the field of natural product and small molecule research.},
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The electron cryo-microscopy (cryo-EM) method Microcrystal Electron Diffraction (MicroED) allows the collection of high-resolution structural data from vanishingly small crystals that appear like amorphous powders or very fine needles. Since its debut in 2013, data collection and analysis schemes have been fine-tuned, and there are currently close to 100 structures determined by MicroED. Although originally developed to study proteins, MicroED is also very powerful for smaller systems, with some recent and very promising examples from the field of natural products. Herein, we review what has been achieved so far and provide examples of natural product structures, as well as demonstrate the expected future impact of MicroED to the field of natural product and small molecule research.
Halaby, Steve; Martynowycz, Michael W.; Zhu, Ziyue; Tretiak, Sergei; Zhugayevych, Andriy; Gonen, Tamir; and Martin Seifrid,
Microcrystal Electron Diffraction for Molecular Design of Functional Non-Fullerene Acceptor Structures Journal Article
In: Chem. Mater., 33 (3), pp. 966–977, 2021.
@article{2021_Halaby,
title = {Microcrystal Electron Diffraction for Molecular Design of Functional Non-Fullerene Acceptor Structures},
author = {Steve Halaby and Michael W. Martynowycz and Ziyue Zhu and Sergei Tretiak and Andriy Zhugayevych and Tamir Gonen and and Martin Seifrid},
doi = {10.1021/acs.chemmater.0c04111},
year = {2021},
date = {2021-01-25},
journal = {Chem. Mater.},
volume = {33},
number = {3},
pages = {966--977},
abstract = {Understanding the relationship between molecular structure and solid-state arrangement informs about the design of new organic semiconductor (OSC) materials with improved optoelectronic properties. However, determining their atomic structure remains challenging. Here, we report the lattice organization of two non-fullerene acceptors (NFAs) determined using microcrystal electron diffraction (MicroED) from crystals not tractable by X-ray crystallography. The MicroED structure of o-IDTBR was determined from a powder without crystallization, and a new polymorph of ITIC-Th is identified with the most distorted backbone of any NFA. Electronic structure calculations elucidate the relationships between molecular structures, lattice arrangements, and charge-transport properties for a number of NFA lattices. The high dimensionality of the connectivity of the 3D wire mesh topology is the best for robust charge transport within NFA crystals. However, some examples suffer from uneven electronic coupling. MicroED combined with advanced electronic structure modeling is a powerful new approach for structure determination, exploring polymorphism and guiding the design of new OSCs and NFAs.},
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Understanding the relationship between molecular structure and solid-state arrangement informs about the design of new organic semiconductor (OSC) materials with improved optoelectronic properties. However, determining their atomic structure remains challenging. Here, we report the lattice organization of two non-fullerene acceptors (NFAs) determined using microcrystal electron diffraction (MicroED) from crystals not tractable by X-ray crystallography. The MicroED structure of o-IDTBR was determined from a powder without crystallization, and a new polymorph of ITIC-Th is identified with the most distorted backbone of any NFA. Electronic structure calculations elucidate the relationships between molecular structures, lattice arrangements, and charge-transport properties for a number of NFA lattices. The high dimensionality of the connectivity of the 3D wire mesh topology is the best for robust charge transport within NFA crystals. However, some examples suffer from uneven electronic coupling. MicroED combined with advanced electronic structure modeling is a powerful new approach for structure determination, exploring polymorphism and guiding the design of new OSCs and NFAs.
Martynowycz, Michael W.; Gonen, Tamir
Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking Journal Article
In: Structure, 29 (1), pp. 88–95.e2, 2021.
@article{2020a_Martynowycz,
title = {Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking},
author = {Michael W. Martynowycz and Tamir Gonen},
doi = {10.1016/j.str.2020.09.003},
year = {2021},
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journal = {Structure},
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abstract = {A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of approximately 200 nm using a focused ion beam, and MicroED data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Both the number of ligands bound and their occupancy is higher using on-grid soaking of microcrystals compared with much larger crystals treated similarly and investigated by X-ray crystallography. These results indicate that on-grid soaking ligands into microcrystals results in efficient uptake of ligands into protein microcrystals.},
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A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of approximately 200 nm using a focused ion beam, and MicroED data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Both the number of ligands bound and their occupancy is higher using on-grid soaking of microcrystals compared with much larger crystals treated similarly and investigated by X-ray crystallography. These results indicate that on-grid soaking ligands into microcrystals results in efficient uptake of ligands into protein microcrystals.
Gonen, Tamir; Nannenga, Brent (Ed.)
cryoEM: Methods and Protocols Book
Springer US, 2021, ISBN: 978-1-0716-0965-1.
@book{GonenNannenga2021,
title = {cryoEM: Methods and Protocols},
editor = {Tamir Gonen and Brent Nannenga},
doi = {10.1007/978-1-0716-0966-8},
isbn = {978-1-0716-0965-1},
year = {2021},
date = {2021-00-00},
volume = {2215},
publisher = {Springer US},
series = {Methods in Molecular Biology},
abstract = {This volume details the most up-to-date cryo-EM techniques from leading researchers. Chapters are organized into four parts with emphasis on electron cryotomography, single particle analysis, and the crystal based cryo-EM methods of 2D electron crystallography, and MicroED for the study of 3D crystals. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, CryoEM: Methods and Protocols aims to serve as an excellent resource on cryo-EM and can serve as the foundation for new researchers to this growing field in structural biology. },
keywords = {},
pubstate = {published},
tppubtype = {book}
}
This volume details the most up-to-date cryo-EM techniques from leading researchers. Chapters are organized into four parts with emphasis on electron cryotomography, single particle analysis, and the crystal based cryo-EM methods of 2D electron crystallography, and MicroED for the study of 3D crystals. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, CryoEM: Methods and Protocols aims to serve as an excellent resource on cryo-EM and can serve as the foundation for new researchers to this growing field in structural biology.
Authoritative and cutting-edge, CryoEM: Methods and Protocols aims to serve as an excellent resource on cryo-EM and can serve as the foundation for new researchers to this growing field in structural biology.
2020
Martynowycz, M W; Khan, F; Hattne, J; Abramson, J; Gonen, T
MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel Journal Article
In: Proc Natl Acad Sci U S A, 117 (51), pp. 32380–32385, 2020.
@article{pmid33293416,
title = {MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel},
author = {M W Martynowycz and F Khan and J Hattne and J Abramson and T Gonen},
doi = {10.1073/pnas.2020010117},
year = {2020},
date = {2020-12-22},
journal = {Proc Natl Acad Sci U S A},
volume = {117},
number = {51},
pages = {32380--32385},
abstract = {A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.
Zhu, Lan; Bu, Guanhong; Jing, Liang; Shi, Dan; Lee, Ming-Yue; Gonen, Tamir; Liu, Wei; Nannenga, Brent L.
Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED Journal Article
In: Structure, 28 (10), pp. 1149–1159.e4, 2020.
@article{2020_Zhu,
title = {Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED},
author = {Lan Zhu and Guanhong Bu and Liang Jing and Dan Shi and Ming-Yue Lee and Tamir Gonen and Wei Liu and Brent L. Nannenga},
url = {https://cryoem.ucla.edu/wp-content/uploads/2020_Zhu.pdf, Main text},
doi = {https://doi.org/10.1016/j.str.2020.07.006},
year = {2020},
date = {2020-10-06},
journal = {Structure},
volume = {28},
number = {10},
pages = {1149--1159.e4},
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. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy 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 converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A2A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy 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 converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A2A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.
Richards, Logan S.; Millán, Claudia; Miao, Jennifer; Martynowycz, Michael W.; Sawaya, Michael R.; Gonen, Tamir; Borges, Rafael J.; Usón, Isabel; Rodriguez, Jose A.
Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER Journal Article
In: Acta Crystallogr D Biol Crystallogr., 76 (8), pp. 703-712, 2020.
@article{2020_Richards,
title = {Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER},
author = {Logan S. Richards and Claudia Millán and Jennifer Miao and Michael W. Martynowycz and Michael R. Sawaya and Tamir Gonen and Rafael J. Borges and Isabel Usón and Jose A. Rodriguez},
url = {https://cryoem.ucla.edu/wp-content/uploads/2020_Richards.pdf, Main text},
doi = {10.1107/S2059798320008049},
year = {2020},
date = {2020-07-27},
journal = {Acta Crystallogr D Biol Crystallogr.},
volume = {76},
number = {8},
pages = {703-712},
abstract = {Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose.
Nguyen, Chi; Gonen, Tamir
Beyond protein structure determination with MicroED Journal Article
In: Curr. Opin. Struct. Biol., 64 , pp. 51–58, 2020.
@article{2020_Nguyen,
title = {Beyond protein structure determination with MicroED},
author = {Chi Nguyen and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2020_NguyenGonen.pdf, Main text},
doi = {10.1016/j.sbi.2020.05.018},
year = {2020},
date = {2020-06-28},
urldate = {2020-06-28},
journal = {Curr. Opin. Struct. Biol.},
volume = {64},
pages = {51–58},
abstract = {Microcrystal electron diffraction (MicroED) was first coined and developed in 2013 at the Janelia Research Campus as a new modality in electron cryomicroscopy (cryoEM). Since then, MicroED has not only made important contributions in pushing the resolution limits of cryoEM protein structure characterization but also of peptides, small-organic and inorganic molecules, and natural-products that have resisted structure determination by other methods. This review showcases important recent developments in MicroED, highlighting the importance of the technique in fields of studies beyond protein structure determination where MicroED is beginning to have paradigm shifting roles.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) was first coined and developed in 2013 at the Janelia Research Campus as a new modality in electron cryomicroscopy (cryoEM). Since then, MicroED has not only made important contributions in pushing the resolution limits of cryoEM protein structure characterization but also of peptides, small-organic and inorganic molecules, and natural-products that have resisted structure determination by other methods. This review showcases important recent developments in MicroED, highlighting the importance of the technique in fields of studies beyond protein structure determination where MicroED is beginning to have paradigm shifting roles.
Martynowycz, Michael W.; Gonen, Tamir
Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking Online
bioRxiv 2020.
@online{2020b_Martynowycz,
title = {Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking},
author = {Martynowycz, Michael W. and Gonen, Tamir},
doi = {10.1101/2020.05.25.115246},
year = {2020},
date = {2020-05-26},
journal = {bioR},
organization = {bioRxiv},
abstract = {A method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoEM vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of 200nm using a focused ion-beam, and microcrystal electron diffraction (MicroED) data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Compared to much larger crystals investigated by X-ray crystallography, both the number of ligands bound and their occupancy was higher in MicroED. These results indicate that soaking ligands into microcrystals in this way results in a more efficient uptake than in larger crystals that are typically used in drug discovery pipelines by X-ray crystallography.
},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
A method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoEM vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of 200nm using a focused ion-beam, and microcrystal electron diffraction (MicroED) data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Compared to much larger crystals investigated by X-ray crystallography, both the number of ligands bound and their occupancy was higher in MicroED. These results indicate that soaking ligands into microcrystals in this way results in a more efficient uptake than in larger crystals that are typically used in drug discovery pipelines by X-ray crystallography.
Wolff, A M; Young, I D; Sierra, R G; Brewster, A S; Martynowycz, M W; Nango, E; Sugahara, M; Nakane, T; Ito, K; Aquila, A; Bhowmick, A; Biel, J T; Carbajo, S; Cohen, A E; Cortez, S; Gonzalez, A; Hino, T; Im, D; Koralek, J D; Kubo, M; Lazarou, T S; Nomura, T; Owada, S; Samelson, A J; Tanaka, T; Tanaka, R; Thompson, E M; van den Bedem, H; Woldeyes, R A; Yumoto, F; Zhao, W; Tono, K; Boutet, S; Iwata, S; Gonen, T; Sauter, N K; Fraser, J S; Thompson, M C
In: IUCrJ, 7 (Pt 2), pp. 306–323, 2020.
@article{pmid32148858,
title = {Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals},
author = {A M Wolff and I D Young and R G Sierra and A S Brewster and M W Martynowycz and E Nango and M Sugahara and T Nakane and K Ito and A Aquila and A Bhowmick and J T Biel and S Carbajo and A E Cohen and S Cortez and A Gonzalez and T Hino and D Im and J D Koralek and M Kubo and T S Lazarou and T Nomura and S Owada and A J Samelson and T Tanaka and R Tanaka and E M Thompson and H van den Bedem and R A Woldeyes and F Yumoto and W Zhao and K Tono and S Boutet and S Iwata and T Gonen and N K Sauter and J S Fraser and M C Thompson},
doi = {10.1107/S205225252000072X},
year = {2020},
date = {2020-03-01},
journal = {IUCrJ},
volume = {7},
number = {Pt 2},
pages = {306--323},
abstract = {Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.
Martynowycz, M W; Hattne, J; Gonen, T
Experimental Phasing of MicroED Data Using Radiation Damage Journal Article
In: Structure, 28 (4), pp. 458–464, 2020.
@article{pmid32023481,
title = {Experimental Phasing of MicroED Data Using Radiation Damage},
author = {M W Martynowycz and J Hattne and T Gonen},
doi = {10.1016/j.str.2020.01.008},
year = {2020},
date = {2020-01-01},
journal = {Structure},
volume = {28},
number = {4},
pages = {458--464},
abstract = {We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present an example of an experimentally phased structure using only MicroED data. The structure of a seven-residue peptide is solved starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous-rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a "low-dose dataset." The data from the second pass were similarly merged to form a "damaged dataset." Differences between these two datasets were used to identify a single heavy-atom site from a Patterson difference map, and initial phases were generated. Finally, the structure was completed by iterative cycles of modeling and refinement.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present an example of an experimentally phased structure using only MicroED data. The structure of a seven-residue peptide is solved starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous-rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a "low-dose dataset." The data from the second pass were similarly merged to form a "damaged dataset." Differences between these two datasets were used to identify a single heavy-atom site from a Patterson difference map, and initial phases were generated. Finally, the structure was completed by iterative cycles of modeling and refinement.
2019
Dick, Markus; Sarai, Nicholas S; Martynowycz, Michael W; Gonen, Tamir; Arnold, Frances H
Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation Journal Article
In: J. Am. Chem. Soc., 2019.
@article{pmid31747522,
title = {Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation},
author = {Markus Dick and Nicholas S Sarai and Michael W Martynowycz and Tamir Gonen and Frances H Arnold},
doi = {10.1021/jacs.9b09864},
year = {2019},
date = {2019-11-01},
journal = {J. Am. Chem. Soc.},
abstract = {We previously engineered the tryptophan synthase β-subunit (TrpB), which catalyzes the condensation of L-serine and indole to L-tryptophan, to synthesize a range of noncanonical amino acids from L-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds by attacking N1 of the substrate. Protecting N1 encouraged evolution towards C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pfquat with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereo-center at the β-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the β-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pfquat offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We previously engineered the tryptophan synthase β-subunit (TrpB), which catalyzes the condensation of L-serine and indole to L-tryptophan, to synthesize a range of noncanonical amino acids from L-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds by attacking N1 of the substrate. Protecting N1 encouraged evolution towards C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pfquat with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereo-center at the β-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the β-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pfquat offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.
Martynowycz, Michael W; Zhao, Wei; Hattne, Johan; Jensen, Grant J; Gonen, Tamir
Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED Journal Article
In: Structure, 27 (10), pp. 1594–1600, 2019.
@article{pmid31422911,
title = {Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED},
author = {Michael W Martynowycz and Wei Zhao and Johan Hattne and Grant J Jensen and Tamir Gonen},
doi = {10.1016/j.str.2019.07.004},
year = {2019},
date = {2019-10-01},
journal = {Structure},
volume = {27},
number = {10},
pages = {1594--1600},
abstract = {Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.
Hattne, Johan; Martynowycz, Michael W; Penczek, Pawel A; Gonen, Tamir
MicroED with the Falcon III direct electron detector Journal Article
In: IUCrJ, 6 (Pt 5), pp. 921–926, 2019.
@article{pmid31576224,
title = {MicroED with the Falcon III direct electron detector},
author = {Johan Hattne and Michael W Martynowycz and Pawel A Penczek and Tamir Gonen},
doi = {10.1107/S2052252519010583},
year = {2019},
date = {2019-09-01},
journal = {IUCrJ},
volume = {6},
number = {Pt 5},
pages = {921--926},
abstract = {Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.
Warmack, Rebeccah A; Boyer, David R; Zee, Chih-Te; Richards, Logan S; Sawaya, Michael R; Cascio, Duilio; Gonen, Tamir; Eisenberg, David S; Clarke, Steven G
Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface Journal Article
In: Nat Commun, 10 (1), pp. 3357, 2019.
@article{pmid31350392,
title = {Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface},
author = {Rebeccah A Warmack and David R Boyer and Chih-Te Zee and Logan S Richards and Michael R Sawaya and Duilio Cascio and Tamir Gonen and David S Eisenberg and Steven G Clarke},
doi = {10.1038/s41467-019-11183-z},
year = {2019},
date = {2019-07-26},
journal = {Nat Commun},
volume = {10},
number = {1},
pages = {3357},
abstract = {Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.
Ting, Chi P; Funk, Michael A; Halaby, Steve L; Zhang, Zhengan; Gonen, Tamir; van der Donk, Wilfred A
Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products Journal Article
In: Science, 365 (6450), pp. 280–284, 2019.
@article{pmid31320540,
title = {Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products},
author = {Chi P Ting and Michael A Funk and Steve L Halaby and Zhengan Zhang and Tamir Gonen and Wilfred A van der Donk},
doi = {10.1126/science.aau6232},
year = {2019},
date = {2019-07-19},
journal = {Science},
volume = {365},
number = {6450},
pages = {280--284},
abstract = {Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA-dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid-derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid-derived natural products.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA-dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid-derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid-derived natural products.
Martynowycz, Michael W.; Gonen, Tamir
MicroED Structure of Hexagonal Ice Ih Online
ChemRxiv 2019.
@online{2019_martynowycz,
title = {MicroED Structure of Hexagonal Ice Ih},
author = {Martynowycz, Michael W. and Gonen, Tamir},
doi = {10.26434/chemrxiv.8298641.v1},
year = {2019},
date = {2019-06-20},
organization = {ChemRxiv},
abstract = {The structure of ice Ih is solved from a single nanocrystal to a resolution of 0.53Å using the cryoEM method microcrystal electron diffraction (MicroED). Data were collected at just above liquid nitrogen temperatures (~80K) in ultra-high vacuum (~8 x 10-7 Pa) using a total exposure of less than 1e- Å-2. The model has the same unit cell dimensions and space group as structures previously determined by both X-ray and neutron scattering of ice Ih, and obeys the Bernal–Fowler ice rules. Both axial and distal hydrogen densities between oxygen atoms of non-deuterated water ice are resolved. Unaccounted for density between axial hydrogen atoms is observed, and may be a direct observation of a polar bond caused by the electric dipole between oxygen and hydrogen atoms. These observations may have implications for the effects of electron radiation on non-terrestrial ice formations because the conditions experienced by the sample in these experiments are mimetic to those found in near solar space or some planetary bodies without atmospheres, where water ice deposits are exposed to high-energy cosmic rays, thermal stellar emissions, and radiation stemming from solar flares.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
The structure of ice Ih is solved from a single nanocrystal to a resolution of 0.53Å using the cryoEM method microcrystal electron diffraction (MicroED). Data were collected at just above liquid nitrogen temperatures (~80K) in ultra-high vacuum (~8 x 10-7 Pa) using a total exposure of less than 1e- Å-2. The model has the same unit cell dimensions and space group as structures previously determined by both X-ray and neutron scattering of ice Ih, and obeys the Bernal–Fowler ice rules. Both axial and distal hydrogen densities between oxygen atoms of non-deuterated water ice are resolved. Unaccounted for density between axial hydrogen atoms is observed, and may be a direct observation of a polar bond caused by the electric dipole between oxygen and hydrogen atoms. These observations may have implications for the effects of electron radiation on non-terrestrial ice formations because the conditions experienced by the sample in these experiments are mimetic to those found in near solar space or some planetary bodies without atmospheres, where water ice deposits are exposed to high-energy cosmic rays, thermal stellar emissions, and radiation stemming from solar flares.
de la Cruz, Jason M; Martynowycz, Michael W; Hattne, Johan; Gonen, Tamir
MicroED data collection with SerialEM Journal Article
In: Ultramicroscopy, 201 , pp. 77–80, 2019.
@article{pmid30986656,
title = {MicroED data collection with SerialEM},
author = {Jason M de la Cruz and Michael W Martynowycz and Johan Hattne and Tamir Gonen},
doi = {10.1016/j.ultramic.2019.03.009},
year = {2019},
date = {2019-06-01},
journal = {Ultramicroscopy},
volume = {201},
pages = {77--80},
abstract = {The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.
Nannenga, Brent L; Gonen, Tamir
The cryo-EM method microcrystal electron diffraction (MicroED) Journal Article
In: Nat. Methods, 16 (5), pp. 369–379, 2019.
@article{pmid31040436,
title = {The cryo-EM method microcrystal electron diffraction (MicroED)},
author = {Brent L Nannenga and Tamir Gonen},
doi = {10.1038/s41592-019-0395-x},
year = {2019},
date = {2019-04-29},
journal = {Nat. Methods},
volume = {16},
number = {5},
pages = {369--379},
abstract = {In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users.
Martynowycz, Michael W; Zhao, Wei; Hattne, Johan; Jensen, Grant J; Gonen, Tamir
Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size Journal Article
In: Structure, 27 (3), pp. 545–548, 2019.
@article{pmid30661853,
title = {Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size},
author = {Michael W Martynowycz and Wei Zhao and Johan Hattne and Grant J Jensen and Tamir Gonen},
doi = {10.1016/j.str.2018.12.003},
year = {2019},
date = {2019-03-05},
journal = {Structure},
volume = {27},
number = {3},
pages = {545--548},
abstract = {Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.
Zee, Chih-Te; Glynn, Calina; Gallagher-Jones, Marcus; Miao, Jennifer; Santiago, Carlos G; Cascio, Duilio; Gonen, Tamir; Sawaya, Michael R; Rodriguez, Jose A
Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ Journal Article
In: IUCrJ, 6 (Pt 2), pp. 197–205, 2019.
@article{pmid30867917,
title = {Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ},
author = {Chih-Te Zee and Calina Glynn and Marcus Gallagher-Jones and Jennifer Miao and Carlos G Santiago and Duilio Cascio and Tamir Gonen and Michael R Sawaya and Jose A Rodriguez},
doi = {10.1107/S2052252518017621},
year = {2019},
date = {2019-03-01},
journal = {IUCrJ},
volume = {6},
number = {Pt 2},
pages = {197--205},
abstract = {The ice-nucleation protein InaZ from Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. Ab initio structures of this repeat segment, resolved to 1.1 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 Journal Article
In: Proc. Natl. Acad. Sci. U.S.A., 116 (6), pp. 2086–2090, 2019.
@article{pmid30659158,
title = {Structural basis for substrate binding and specificity of a sodium-alanine symporter AgcS},
author = {Jinming Ma and Hsiang-Ting Lei and Francis E Reyes and Silvia Sanchez-Martinez and Maen F Sarhan and Johan Hattne and Tamir Gonen},
doi = {10.1073/pnas.1806206116},
year = {2019},
date = {2019-01-18},
journal = {Proc. Natl. Acad. Sci. U.S.A.},
volume = {116},
number = {6},
pages = {2086--2090},
abstract = {The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.
Griner, Sarah L; Seidler, Paul; Bowler, Jeannette; Murray, Kevin A; Yang, Tianxiao Peter; Sahay, Shruti; Sawaya, Michael R; Cascio, Duilio; Rodriguez, Jose A; Philipp, Stephan; Sosna, Justyna; Glabe, Charles G; Gonen, Tamir; Eisenberg, David S
Structure-based inhibitors of amyloid beta core suggest a common interface with tau Journal Article
In: Elife, 8 , 2019.
@article{pmid31612856,
title = {Structure-based inhibitors of amyloid beta core suggest a common interface with tau},
author = {Sarah L Griner and Paul Seidler and Jeannette Bowler and Kevin A Murray and Tianxiao Peter Yang and Shruti Sahay and Michael R Sawaya and Duilio Cascio and Jose A Rodriguez and Stephan Philipp and Justyna Sosna and Charles G Glabe and Tamir Gonen and David S Eisenberg},
doi = {10.7554/eLife.46924.001},
year = {2019},
date = {2019-01-01},
journal = {Elife},
volume = {8},
abstract = {Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline.
2018
Purdy, Michael D; Shi, Dan; Chrustowicz, Jakub; Hattne, Johan; Gonen, Tamir; Yeager, Mark
MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat Journal Article
In: Proc. Natl. Acad. Sci. U.S.A., 115 (52), pp. 13258–13263, 2018.
@article{pmid30530702,
title = {MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat},
author = {Michael D Purdy and Dan Shi and Jakub Chrustowicz and Johan Hattne and Tamir Gonen and Mark Yeager},
doi = {10.1073/pnas.1806806115},
year = {2018},
date = {2018-12-26},
journal = {Proc. Natl. Acad. Sci. U.S.A.},
volume = {115},
number = {52},
pages = {13258--13263},
abstract = {HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.
Jones, Christopher G; Martynowycz, Michael W; Hattne, Johan; Fulton, Tyler J; Stoltz, Brian M; Rodriguez, Jose A; Nelson, Hosea M; Gonen, Tamir
The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination Journal Article
In: ACS Cent Sci, 4 (11), pp. 1587–1592, 2018.
@article{pmid30555912,
title = {The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination},
author = {Christopher G Jones and Michael W Martynowycz and Johan Hattne and Tyler J Fulton and Brian M Stoltz and Jose A Rodriguez and Hosea M Nelson and Tamir Gonen},
doi = {10.1021/acscentsci.8b00760},
year = {2018},
date = {2018-11-02},
journal = {ACS Cent Sci},
volume = {4},
number = {11},
pages = {1587--1592},
abstract = {In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule's structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10-15 g) resulting in atomic resolution (<1 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.
Martynowycz, Michael W.; Zhao, Wei; Hattne, Johen; Jensen, Grant J.; Gonen, Tamir
Collection of continuous rotation MicroED Data from Ion Beam Milled Crystals of Any Size Online
bioRxiv 2018.
@online{2018_martynowycz,
title = {Collection of continuous rotation MicroED Data from Ion Beam Milled Crystals of Any Size},
author = {Martynowycz, Michael W. and Zhao, Wei and Hattne, Johen and Jensen, Grant J. and Gonen, Tamir},
doi = {10.1101/425611},
year = {2018},
date = {2018-09-24},
organization = {bioRxiv},
abstract = {Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoEM as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to ideal dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoEM as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to ideal dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.
Ma, Jinming; Lei, Hsiang-Ting; Gonen, Tamir
A conformational change in the N terminus of SLC38A9 signals mTORC1 activation Online
bioRxiv 2018.
@online{2018_ma_b,
title = {A conformational change in the N terminus of SLC38A9 signals mTORC1 activation},
author = {Ma, Jinming and Lei, Hsiang-Ting and Gonen, Tamir},
doi = {10.1101/339937},
year = {2018},
date = {2018-06-06},
journal = {bio},
organization = {bioRxiv},
abstract = {mTORC1 is a central signal hub that integrates multiple environmental cues, such as cellular stresses, energy levels, nutrients and certain amino acids, to modulate metabolic status and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, has emerged as a sensor for luminal arginine levels and as an activator of mTOCRC1. The activation of mTORC1 occurs through the N-terminal domain of SLC38A9. Here, we determined the crystal structure of SLC38A9 and surprisingly found its N-terminal fragment inserted deep into the transporter, bound in the substrate binding pocket where normally arginine would bind. Compared with our recent arginine bound structure of SLC38A9, a significant conformational change of the N-terminal domain was observed. A ball-and-chain model is proposed for mTORC1 activation where in the starved state the N-terminal domain of SLC38A9 is buried deep in the transporter but in the fed state the N-terminal domain could be released becoming free to bind the Rag GTPase complex and to activate mTORC1. This work provides important new insights into how SLC38A9 senses the fed state and activates the mTORC1 pathways in response to dietary amino acids.
},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
mTORC1 is a central signal hub that integrates multiple environmental cues, such as cellular stresses, energy levels, nutrients and certain amino acids, to modulate metabolic status and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, has emerged as a sensor for luminal arginine levels and as an activator of mTOCRC1. The activation of mTORC1 occurs through the N-terminal domain of SLC38A9. Here, we determined the crystal structure of SLC38A9 and surprisingly found its N-terminal fragment inserted deep into the transporter, bound in the substrate binding pocket where normally arginine would bind. Compared with our recent arginine bound structure of SLC38A9, a significant conformational change of the N-terminal domain was observed. A ball-and-chain model is proposed for mTORC1 activation where in the starved state the N-terminal domain of SLC38A9 is buried deep in the transporter but in the fed state the N-terminal domain could be released becoming free to bind the Rag GTPase complex and to activate mTORC1. This work provides important new insights into how SLC38A9 senses the fed state and activates the mTORC1 pathways in response to dietary amino acids.
Lei, Hsiang-Ting; Ma, Jinming; Martinez, Silvia Sanchez; Gonen, Tamir
Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state Journal Article
In: Nat. Struct. Mol. Biol., 25 (6), pp. 522–527, 2018.
@article{pmid29872228,
title = {Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state},
author = {Hsiang-Ting Lei and Jinming Ma and Silvia Sanchez Martinez and Tamir Gonen},
doi = {10.1038/s41594-018-0072-2},
year = {2018},
date = {2018-06-05},
journal = {Nat. Struct. Mol. Biol.},
volume = {25},
number = {6},
pages = {522--527},
abstract = {Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.
Liu, Shian; Gonen, Tamir
MicroED structure of the NaK ion channel reveals a Na⁺ partition process into the selectivity filter Journal Article
In: Commun Biol, 1 , pp. 38, 2018.
@article{pmid30167468,
title = {MicroED structure of the NaK ion channel reveals a Na⁺ partition process into the selectivity filter},
author = {Shian Liu and Tamir Gonen},
doi = {10.1038/s42003-018-0040-8},
year = {2018},
date = {2018-05-03},
journal = {Commun Biol},
volume = {1},
pages = {38},
abstract = {Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.
Hattne, Johan; Shi, Dan; Glynn, Calina; Zee, Chih-Te; Gallagher-Jones, Marcus; Martynowycz, Michael W; Rodriguez, Jose A; Gonen, Tamir
Analysis of Global and Site-Specific Radiation Damage in Cryo-EM Journal Article
In: Structure, 26 (5), pp. 759–766, 2018.
@article{pmid29706530,
title = {Analysis of Global and Site-Specific Radiation Damage in Cryo-EM},
author = {Johan Hattne and Dan Shi and Calina Glynn and Chih-Te Zee and Marcus Gallagher-Jones and Michael W Martynowycz and Jose A Rodriguez and Tamir Gonen},
doi = {10.1016/j.str.2018.03.021},
year = {2018},
date = {2018-05-01},
journal = {Structure},
volume = {26},
number = {5},
pages = {759--766},
abstract = {Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.
Ma, Jinming; Lei, Hsiang-Ting; Reyes, Francis E.; Sanchez-Martinez, Silvia; Sarhan, Maen; Gonen, Tamir
Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS Online
bioRxiv 2018.
@online{2018_ma,
title = {Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS},
author = {Ma, Jinming and Lei, Hsiang-Ting and Reyes, Francis E. and Sanchez-Martinez, Silvia and Sarhan, Maen and Gonen, Tamir},
doi = {10.1101/293811},
year = {2018},
date = {2018-04-03},
organization = {bioRxiv},
abstract = {The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.
Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Yeates, Todd O
Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system Journal Article
In: Proc. Natl. Acad. Sci. U.S.A., 115 (13), pp. 3362–3367, 2018.
@article{pmid29507202,
title = {Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system},
author = {Yuxi Liu and Shane Gonen and Tamir Gonen and Todd O Yeates},
doi = {10.1073/pnas.1718825115},
year = {2018},
date = {2018-03-27},
journal = {Proc. Natl. Acad. Sci. U.S.A.},
volume = {115},
number = {13},
pages = {3362--3367},
abstract = {Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.
Guenther, Elizabeth L; Ge, Peng; Trinh, Hamilton; Sawaya, Michael R; Cascio, Duilio; Boyer, David R; Gonen, Tamir; Zhou, Hong Z; Eisenberg, David S
Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2 Journal Article
In: Nat. Struct. Mol. Biol., 25 (4), pp. 311–319, 2018.
@article{pmid29531287,
title = {Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2},
author = {Elizabeth L Guenther and Peng Ge and Hamilton Trinh and Michael R Sawaya and Duilio Cascio and David R Boyer and Tamir Gonen and Hong Z Zhou and David S Eisenberg},
doi = {10.1038/s41594-018-0045-5},
year = {2018},
date = {2018-03-12},
journal = {Nat. Struct. Mol. Biol.},
volume = {25},
number = {4},
pages = {311--319},
abstract = {Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.
Martynowycz, Michael W; Gonen, Tamir
From electron crystallography of 2D crystals to MicroED of 3D crystals Journal Article
In: Curr Opin Colloid Interface Sci, 34 , pp. 9–16, 2018.
@article{pmid30166936,
title = {From electron crystallography of 2D crystals to MicroED of 3D crystals},
author = {Michael W Martynowycz and Tamir Gonen},
doi = {10.1016/j.cocis.2018.01.010},
year = {2018},
date = {2018-03-01},
journal = {Curr Opin Colloid Interface Sci},
volume = {34},
pages = {9--16},
abstract = {Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.
Hughes, Michael P; Sawaya, Michael R; Boyer, David R; Goldschmidt, Lukasz; Rodriguez, Jose A; Cascio, Duilio; Chong, Lisa; Gonen, Tamir; Eisenberg, David S
Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks Journal Article
In: Science, 359 (6376), pp. 698–701, 2018.
@article{pmid29439243,
title = {Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks},
author = {Michael P Hughes and Michael R Sawaya and David R Boyer and Lukasz Goldschmidt and Jose A Rodriguez and Duilio Cascio and Lisa Chong and Tamir Gonen and David S Eisenberg},
doi = {10.1126/science.aan6398},
year = {2018},
date = {2018-02-09},
journal = {Science},
volume = {359},
number = {6376},
pages = {698--701},
abstract = {Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.
Nannenga, Brent L; Gonen, Tamir
MicroED: a versatile cryoEM method for structure determination Journal Article
In: Emerg Top Life Sci, 2 (1), pp. 1–8, 2018.
@article{pmid30167465,
title = {MicroED: a versatile cryoEM method for structure determination},
author = {Brent L Nannenga and Tamir Gonen},
doi = {10.1042/ETLS20170082},
year = {2018},
date = {2018-02-06},
journal = {Emerg Top Life Sci},
volume = {2},
number = {1},
pages = {1--8},
abstract = {Micro-electron diffraction, or MicroED, is a structure determination method that uses a cryo-transmission electron microscope to collect electron diffraction data from nanocrystals. This technique has been successfully used to determine the high-resolution structures of many targets from crystals orders of magnitude smaller than what is needed for X-ray diffraction experiments. In this review, we will describe the MicroED method and recent structures that have been determined. Additionally, applications of electron diffraction to the fields of small molecule crystallography and materials science will be discussed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Micro-electron diffraction, or MicroED, is a structure determination method that uses a cryo-transmission electron microscope to collect electron diffraction data from nanocrystals. This technique has been successfully used to determine the high-resolution structures of many targets from crystals orders of magnitude smaller than what is needed for X-ray diffraction experiments. In this review, we will describe the MicroED method and recent structures that have been determined. Additionally, applications of electron diffraction to the fields of small molecule crystallography and materials science will be discussed.
Gallagher-Jones, Marcus; Glynn, Calina; Boyer, David R; Martynowycz, Michael W; Hernandez, Evelyn; Miao, Jennifer; Zee, Chih-Te; Novikova, Irina V; Goldschmidt, Lukasz; McFarlane, Heather T; Helguera, Gustavo F; Evans, James E; Sawaya, Michael R; Cascio, Duilio; Eisenberg, David S; Gonen, Tamir; Rodriguez, Jose A
Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp Journal Article
In: Nat. Struct. Mol. Biol., 25 (2), pp. 131–134, 2018.
@article{pmid29335561,
title = {Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp},
author = {Marcus Gallagher-Jones and Calina Glynn and David R Boyer and Michael W Martynowycz and Evelyn Hernandez and Jennifer Miao and Chih-Te Zee and Irina V Novikova and Lukasz Goldschmidt and Heather T McFarlane and Gustavo F Helguera and James E Evans and Michael R Sawaya and Duilio Cascio and David S Eisenberg and Tamir Gonen and Jose A Rodriguez},
doi = {10.1038/s41594-017-0018-0},
year = {2018},
date = {2018-01-15},
journal = {Nat. Struct. Mol. Biol.},
volume = {25},
number = {2},
pages = {131--134},
abstract = {The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-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
Seidler, P M; Boyer, D R; Rodriguez, J A; Sawaya, M R; Cascio, D; Murray, K; Gonen, T; Eisenberg, D S
Structure-based inhibitors of tau aggregation Journal Article
In: Nat Chem, 10 (2), pp. 170–176, 2017.
@article{pmid29359764,
title = {Structure-based inhibitors of tau aggregation},
author = {P M Seidler and D R Boyer and J A Rodriguez and M R Sawaya and D Cascio and K Murray and T Gonen and D S Eisenberg},
doi = {10.1038/nchem.2889},
year = {2017},
date = {2017-11-20},
journal = {Nat Chem},
volume = {10},
number = {2},
pages = {170--176},
abstract = {Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.
Vergara, Sandra; Lukes, Dylan A; Martynowycz, Michael W; Santiago, Ulises; Plascencia-Villa, Germán; Weiss, Simon C; de la Cruz, Jason M; Black, David M; Alvarez, Marcos M; López-Lozano, Xochitl; Barnes, Christopher O; Lin, Guowu; Weissker, Hans-Christian; Whetten, Robert L; Gonen, Tamir; Yacaman, Miguel Jose; Calero, Guillermo
MicroED Structure of Au₁₄₆(p-MBA)₅₇ at Subatomic Resolution Reveals a Twinned FCC Cluster Journal Article
In: J Phys Chem Lett, 8 (22), pp. 5523–5530, 2017.
@article{pmid29072840,
title = {MicroED Structure of Au₁₄₆(p-MBA)₅₇ at Subatomic Resolution Reveals a Twinned FCC Cluster},
author = {Sandra Vergara and Dylan A Lukes and Michael W Martynowycz and Ulises Santiago and Germán Plascencia-Villa and Simon C Weiss and Jason M de la Cruz and David M Black and Marcos M Alvarez and Xochitl López-Lozano and Christopher O Barnes and Guowu Lin and Hans-Christian Weissker and Robert L Whetten and Tamir Gonen and Miguel Jose Yacaman and Guillermo Calero},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_vargara.pdf},
doi = {10.1021/acs.jpclett.7b02621},
year = {2017},
date = {2017-10-26},
journal = {J Phys Chem Lett},
volume = {8},
number = {22},
pages = {5523--5530},
abstract = {Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure, whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure, whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.
Rodriguez, Jose A; Eisenberg, David S; Gonen, Tamir
Taking the measure of MicroED Journal Article
In: Curr. Opin. Struct. Biol., 46 , pp. 79–86, 2017.
@article{pmid28648726,
title = {Taking the measure of MicroED},
author = {Jose A Rodriguez and David S Eisenberg and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_rodriguez.pdf, Main text},
doi = {10.1016/j.sbi.2017.06.004},
year = {2017},
date = {2017-10-01},
journal = {Curr. Opin. Struct. Biol.},
volume = {46},
pages = {79--86},
abstract = {It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.
Wright, Zoë V F; McCarthy, Stephen; Dickman, Rachel; Reyes, Francis E; Sanchez-Martinez, Silvia; Cryar, Adam; Kilford, Iian; Hall, Adrian; Takle, Andrew K; Topf, Maya; Gonen, Tamir; Thalassinos, Konstantinos; Tabor, Alethea B
In: J. Am. Chem. Soc., 139 (37), pp. 13063–13075, 2017.
@article{pmid28880078,
title = {The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Naᵥ1.7},
author = {Zoë V F Wright and Stephen McCarthy and Rachel Dickman and Francis E Reyes and Silvia Sanchez-Martinez and Adam Cryar and Iian Kilford and Adrian Hall and Andrew K Takle and Maya Topf and Tamir Gonen and Konstantinos Thalassinos and Alethea B Tabor},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_wright.pdf, Main text},
doi = {10.1021/jacs.7b06506},
year = {2017},
date = {2017-09-07},
journal = {J. Am. Chem. Soc.},
volume = {139},
number = {37},
pages = {13063--13075},
abstract = {Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Naᵥ1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Naᵥ1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.