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 |
| 2023 | A. L. Patterson award, ACA: The Structural Science Society |
Publications
2023
Danelius, Emma; Porter, Nicholas J.; Unge, Johan; Arnold, Frances H.; Gonen, Tamir
MicroED Structure of a Protoglobin Reactive Carbene Intermediate Journal Article
In: J. Am. Chem. Soc., 2023, ISSN: 1520-5126.
@article{Danelius2023,
title = {MicroED Structure of a Protoglobin Reactive Carbene Intermediate},
author = {Emma Danelius and Nicholas J. Porter and Johan Unge and Frances H. Arnold and Tamir Gonen},
doi = {10.1021/jacs.2c12004},
issn = {1520-5126},
year = {2023},
date = {2023-03-22},
journal = {J. Am. Chem. Soc.},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Martynowycz, Michael W; Shiriaeva, Anna; Clabbers, Max T B; Nicolas, William J; Weaver, Sara J; Hattne, Johan; Gonen, Tamir
A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals Journal Article
In: Nat Commun, vol. 14, iss. 1, no. 1, pp. 1086, 2023, ISSN: 2041-1723.
@article{pmid36841804,
title = {A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals},
author = {Michael W Martynowycz and Anna Shiriaeva and Max T B Clabbers and William J Nicolas and Sara J Weaver and Johan Hattne and Tamir Gonen},
doi = {10.1038/s41467-023-36733-4},
issn = {2041-1723},
year = {2023},
date = {2023-02-25},
urldate = {2023-02-01},
journal = {Nat Commun},
volume = {14},
number = {1},
issue = {1},
pages = {1086},
abstract = {Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.
Danelius, Emma; Patel, Khushboo; Gonzalez, Brenda; Gonen, Tamir
MicroED in drug discovery Journal Article
In: Curr Opin Struct Biol, vol. 79, pp. 102549, 2023, ISSN: 1879-033X.
@article{pmid36821888,
title = {MicroED in drug discovery},
author = {Emma Danelius and Khushboo Patel and Brenda Gonzalez and Tamir Gonen},
doi = {10.1016/j.sbi.2023.102549},
issn = {1879-033X},
year = {2023},
date = {2023-02-01},
journal = {Curr Opin Struct Biol},
volume = {79},
pages = {102549},
abstract = {The cryo-electron microscopy (cryo-EM) method microcrystal electron diffraction (MicroED) was initially described in 2013 and has recently gained attention as an emerging technique for research in drug discovery. As compared to other methods in structural biology, MicroED provides many advantages deriving from the use of nanocrystalline material for the investigations. Here, we review the recent advancements in the field of MicroED and show important examples of small molecule, peptide and protein structures that has contributed to the current development of this method as an important tool for drug discovery.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The cryo-electron microscopy (cryo-EM) method microcrystal electron diffraction (MicroED) was initially described in 2013 and has recently gained attention as an emerging technique for research in drug discovery. As compared to other methods in structural biology, MicroED provides many advantages deriving from the use of nanocrystalline material for the investigations. Here, we review the recent advancements in the field of MicroED and show important examples of small molecule, peptide and protein structures that has contributed to the current development of this method as an important tool for drug discovery.
2022
Clabbers, Max T. B.; Martynowycz, Michael W.; Hattne, Johan; Gonen, Tamir
Hydrogens and hydrogen-bond networks in macromolecular MicroED data Journal Article
In: J Struct Biol X, vol. 6, pp. 100078, 2022.
@article{nokey,
title = {Hydrogens and hydrogen-bond networks in macromolecular MicroED data},
author = {Max T.B. Clabbers and Michael W. Martynowycz and Johan Hattne and Tamir Gonen},
doi = {10.1016/j.yjsbx.2022.100078},
year = {2022},
date = {2022-11-10},
urldate = {2022-11-10},
journal = {J Struct Biol X},
volume = {6},
pages = {100078},
organization = {bioRxiv},
abstract = {Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that the scattered electrons carry information about the charged state of atoms and provide relatively stronger contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for understanding protein structure and function, in particular for drug discovery. However, identification of individual hydrogen atom positions typically requires atomic resolution data, and has thus far remained elusive for macromolecular MicroED. Recently, we presented the ab initio structure of triclinic hen egg-white lysozyme at 0.87 Å resolution. The corresponding data were recorded under low exposure conditions using an electron-counting detector from thin crystalline lamellae. Here, using these subatomic resolution MicroED data, we identified over a third of all hydrogen atom positions based on strong difference peaks, and directly visualize hydrogen bonding interactions and the charged states of residues. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering the hydrogen atoms and hydrogen bonding interactions underlying protein structure and function.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that the scattered electrons carry information about the charged state of atoms and provide relatively stronger contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for understanding protein structure and function, in particular for drug discovery. However, identification of individual hydrogen atom positions typically requires atomic resolution data, and has thus far remained elusive for macromolecular MicroED. Recently, we presented the ab initio structure of triclinic hen egg-white lysozyme at 0.87 Å resolution. The corresponding data were recorded under low exposure conditions using an electron-counting detector from thin crystalline lamellae. Here, using these subatomic resolution MicroED data, we identified over a third of all hydrogen atom positions based on strong difference peaks, and directly visualize hydrogen bonding interactions and the charged states of residues. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering the hydrogen atoms and hydrogen bonding interactions underlying protein structure and function.
Danelius, Emma; Porter, Nicholas J.; Unge, Johan; Arnold, Frances H.; Gonen, Tamir
MicroED structure of a protoglobin reactive carbene intermediate Unpublished
bioRxiv, 2022.
@unpublished{Danelius2022,
title = {MicroED structure of a protoglobin reactive carbene intermediate},
author = {Emma Danelius and Nicholas J. Porter and Johan Unge and Frances H. Arnold and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2022.10.18.512604},
doi = {10.1101/2022.10.18.512604},
year = {2022},
date = {2022-10-18},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Microcrystal electron diffraction (MicroED) is an emerging technique which has shown great potential for describing new chemical and biological molecular structures. [1] Several important structures of small molecules, natural products and peptides have been determined usingab initio methods. [2] However, only a couple of novel protein structures have thus far been derived by MicroED. [3, 4] Taking advantage of recent technological advances including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of anAeropyrum pernix protoglobin (Ape Pgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an alphahelix ofApe Pgb into a dynamic loop making the catalytic active site more readily accessible. After exposing the tiny crystals to substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineeredApe Pgb’s new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enables investigation of structures that were previously beyond reach. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Clabbers, Max T. B.; Martynowycz, Michael W.; Hattne, Johan; Nannenga, Brent L.; Gonen, Tamir
Electron-counting MicroED data with the K2 and K3 direct electron detectors Journal Article
In: J Struct Biol, 2022.
@article{pmid36044956,
title = {Electron-counting MicroED data with the K2 and K3 direct electron detectors},
author = {Max T.B. Clabbers and Michael W. Martynowycz and Johan Hattne and Brent L. Nannenga and Tamir Gonen},
doi = {10.1016/j.jsb.2022.107886},
year = {2022},
date = {2022-08-28},
urldate = {2022-08-28},
journal = {J Struct Biol},
organization = {bioRxiv},
abstract = {Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å. Even though a beam stop was not used in these studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å. Even though a beam stop was not used in these studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.
Martynowycz, Michael W.; Shiriaeva, Anna; Clabbers, Max T. B.; Nicolas, William J.; Weaver, Sara J.; Hattne, Johan; Gonen, Tamir
bioRxiv 2022, visited: 26.07.2022.
@online{Martynowycz2022b,
title = {A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals},
author = {Michael W. Martynowycz and Anna Shiriaeva and Max T. B. Clabbers and William J. Nicolas and Sara J. Weaver and Johan Hattne and Tamir Gonen},
doi = {10.1101/2022.07.26.501628},
year = {2022},
date = {2022-07-26},
urldate = {2022-07-26},
journal = {bioRxiv},
organization = {bioRxiv},
abstract = {Crystallization of membrane proteins, such as G protein-coupled receptors (GPCRs), is challenging and frequently requires the use of lipidic cubic phase (LCP) crystallization methods. These typically yield crystals that are too small for synchrotron X-ray crystallography, but ideally suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, the viscous nature of LCP makes sample preparation challenging. The LCP layer is often too thick for transmission electron microscopy (TEM), and crystals buried in LCP cannot be identified topologically using a focused ion-beam and scanning electron microscope (FIB/SEM). Therefore, the LCP needs to either be converted to the sponge phase or entirely removed from the path of the ion-beam to allow identification and milling of these crystals. Unfortunately, conversion of the LCP to sponge phase can also deteriorate the sample. Methods that avoid LCP conversion are needed. Here, we employ a novel approach using an integrated fluorescence light microscope (iFLM) inside of a FIB/SEM to identify fluorescently labelled crystals embedded deep in a thick LCP layer. The crystals are then targeted using fluorescence microscopy and unconverted LCP is removed directly using a plasma focused ion beam (pFIB). To assess the optimal ion source to prepare biological lamellae, we first characterized the four available gas sources on standard crystals of the serine protease, proteinase K. However, lamellae prepared using either argon and xenon produced the highest quality data and structures. Fluorescently labelled crystals of the human adenosine receptor embedded in thick LCP were placed directly onto EM grids without conversion to the sponge phase. Buried microcrystals were identified using iFLM, and deep lamellae were created using the xenon beam. Continuous rotation MicroED data were collected from the exposed crystalline lamella and the structure was determined using a single crystal. This study outlines a robust approach to identifying and milling LCP grown membrane protein crystals for MicroED using single microcrystals, and demonstrates plasma ion-beam milling as a powerful tool for preparing biological lamellae.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
Crystallization of membrane proteins, such as G protein-coupled receptors (GPCRs), is challenging and frequently requires the use of lipidic cubic phase (LCP) crystallization methods. These typically yield crystals that are too small for synchrotron X-ray crystallography, but ideally suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, the viscous nature of LCP makes sample preparation challenging. The LCP layer is often too thick for transmission electron microscopy (TEM), and crystals buried in LCP cannot be identified topologically using a focused ion-beam and scanning electron microscope (FIB/SEM). Therefore, the LCP needs to either be converted to the sponge phase or entirely removed from the path of the ion-beam to allow identification and milling of these crystals. Unfortunately, conversion of the LCP to sponge phase can also deteriorate the sample. Methods that avoid LCP conversion are needed. Here, we employ a novel approach using an integrated fluorescence light microscope (iFLM) inside of a FIB/SEM to identify fluorescently labelled crystals embedded deep in a thick LCP layer. The crystals are then targeted using fluorescence microscopy and unconverted LCP is removed directly using a plasma focused ion beam (pFIB). To assess the optimal ion source to prepare biological lamellae, we first characterized the four available gas sources on standard crystals of the serine protease, proteinase K. However, lamellae prepared using either argon and xenon produced the highest quality data and structures. Fluorescently labelled crystals of the human adenosine receptor embedded in thick LCP were placed directly onto EM grids without conversion to the sponge phase. Buried microcrystals were identified using iFLM, and deep lamellae were created using the xenon beam. Continuous rotation MicroED data were collected from the exposed crystalline lamella and the structure was determined using a single crystal. This study outlines a robust approach to identifying and milling LCP grown membrane protein crystals for MicroED using single microcrystals, and demonstrates plasma ion-beam milling as a powerful tool for preparing biological lamellae.
Clabbers, Max T. B.; Martynowycz, Michael W.; Hattne, Johan; Nannenga, Brent L.; Gonen, Tamir
Electron-counting MicroED data with the K2 and K3 direct electron detectors Unpublished
bioRxiv, 2022.
@unpublished{Clabbers2022,
title = {Electron-counting MicroED data with the K2 and K3 direct electron detectors},
author = {Max T.B. Clabbers and Michael W. Martynowycz and Johan Hattne and Brent L. Nannenga and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2022.07.04.498775},
doi = {10.1101/2022.07.04.498775},
year = {2022},
date = {2022-07-05},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio . Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å. Even though a beam stop was not used in these studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Martynowycz, Michael W.; Gonen, Tamir
Unlocking the potential of microcrystal electron diffraction Journal Article
In: Physics Today, vol. 75, iss. 6, pp. 38–42, 2022.
@article{Martynowycz2022,
title = {Unlocking the potential of microcrystal electron diffraction},
author = {Michael W. Martynowycz and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2022_Physics_today.pdf, Main text},
doi = {10.1063/PT.3.5019},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {Physics Today},
volume = {75},
issue = {6},
pages = {38--42},
abstract = {Atoms stick together in different ways to make the molecules that compose everything we touch and see. Our bodies are made of cells. Cells, in turn, are made of lipids, proteins, nucleic acids, metabolites, and water. Every one of those molecules is made from the same handful of atoms. But although the components are the same, the molecules differ in how many atoms they have and how those atoms are arranged in space.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Atoms stick together in different ways to make the molecules that compose everything we touch and see. Our bodies are made of cells. Cells, in turn, are made of lipids, proteins, nucleic acids, metabolites, and water. Every one of those molecules is made from the same handful of atoms. But although the components are the same, the molecules differ in how many atoms they have and how those atoms are arranged in space.
Nguyen, Chi; Lei, Hsiang-Ting; Lai, Louis Tung Faat; Gallenito, Marc J.; Matthies, Doreen; Gonen, Tamir
Lipid flipping in the omega-3 fatty-acid transporter Online
bioRxiv 2022, visited: 01.06.2022.
@online{nokey,
title = {Lipid flipping in the omega-3 fatty-acid transporter},
author = {Chi Nguyen and Hsiang-Ting Lei and Louis Tung Faat Lai and Marc J. Gallenito and Doreen Matthies and Tamir Gonen},
doi = {10.1101/2022.05.31.494244},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
organization = {bioRxiv},
abstract = {Mfsd2a is the primary transporter for the docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral, learning, and motor dysfunctions to severe microcephaly. Mfsd2a typically transports long-chain unsaturated fatty-acids, including DHA and α-Linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with two recently determined structures of Mfsd2a the molecular details of how this transporter performs the energetically unfavorable task of translocating and flipping lysolipids across the lipid bilayer remained unclear. Here, we report five single-particle cryo-EM structures of the Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and bound to ALA-LPC at four unique positions along the substrate translocation pathway. These Mfsd2a snapshots detail the Na+-dependent flipping mechanism of the lipid-LPC from outer to inner membrane leaflet during ligand translocation through the Mfsd2a substrate tunnel and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with known disease. Together these results provide a model for omega-3 fatty-acid transport and has the potential for the design of the delivery strategies for amphipathic drugs across the BBB.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
Mfsd2a is the primary transporter for the docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral, learning, and motor dysfunctions to severe microcephaly. Mfsd2a typically transports long-chain unsaturated fatty-acids, including DHA and α-Linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with two recently determined structures of Mfsd2a the molecular details of how this transporter performs the energetically unfavorable task of translocating and flipping lysolipids across the lipid bilayer remained unclear. Here, we report five single-particle cryo-EM structures of the Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and bound to ALA-LPC at four unique positions along the substrate translocation pathway. These Mfsd2a snapshots detail the Na+-dependent flipping mechanism of the lipid-LPC from outer to inner membrane leaflet during ligand translocation through the Mfsd2a substrate tunnel and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with known disease. Together these results provide a model for omega-3 fatty-acid transport and has the potential for the design of the delivery strategies for amphipathic drugs across the BBB.
Martynowycz, Michael W; Clabbers, Max T B; Hattne, Johan; Gonen, Tamir
Ab initio phasing macromolecular structures using electron-counted MicroED data Journal Article
In: Nat Methods, vol. 19, iss. 6, pp. 724–729, 2022, ISSN: 1548-7105.
@article{pmid35637302,
title = {Ab initio phasing macromolecular structures using electron-counted MicroED data},
author = {Michael W Martynowycz and Max T B Clabbers and Johan Hattne and Tamir Gonen},
doi = {10.1038/s41592-022-01485-4},
issn = {1548-7105},
year = {2022},
date = {2022-05-30},
urldate = {2022-05-01},
journal = {Nat Methods},
volume = {19},
issue = {6},
pages = {724--729},
abstract = {Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.
Porter, Nicholas J; Danelius, Emma; Gonen, Tamir; Arnold, Frances H
Biocatalytic Carbene Transfer Using Diazirines Journal Article
In: J Am Chem Soc, 2022, ISSN: 1520-5126.
@article{pmid35561334,
title = {Biocatalytic Carbene Transfer Using Diazirines},
author = {Nicholas J Porter and Emma Danelius and Tamir Gonen and Frances H Arnold},
doi = {10.1021/jacs.2c02723},
issn = {1520-5126},
year = {2022},
date = {2022-05-01},
journal = {J Am Chem Soc},
abstract = {Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the variety of accessible products. To overcome this restriction, we have engineered variants of protoglobin (Pgb) that use diazirines as carbene precursors. While the enhanced stability of diazirines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered Pgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an Pgb variant, determined by microcrystal electron diffraction (MicroED), reveals that evolution has enhanced access to the heme active site to facilitate this new-to-nature catalysis. Using readily prepared aryl diazirines as model substrates, we demonstrate the application of these highly stable carbene precursors in biocatalytic cyclopropanation, N-H insertion, and Si-H insertion reactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the variety of accessible products. To overcome this restriction, we have engineered variants of protoglobin (Pgb) that use diazirines as carbene precursors. While the enhanced stability of diazirines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered Pgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an Pgb variant, determined by microcrystal electron diffraction (MicroED), reveals that evolution has enhanced access to the heme active site to facilitate this new-to-nature catalysis. Using readily prepared aryl diazirines as model substrates, we demonstrate the application of these highly stable carbene precursors in biocatalytic cyclopropanation, N-H insertion, and Si-H insertion reactions.
Clabbers, Max T. B.; Martynowycz, Michael W.; Hattne, Johan; Gonen, Tamir
Hydrogens and hydrogen-bond networks in macromolecular MicroED data Unpublished
bioRxiv, 2022.
@unpublished{Clabbers2022b,
title = {Hydrogens and hydrogen-bond networks in macromolecular MicroED data},
author = {Max T.B. Clabbers and Michael W. Martynowycz and Johan Hattne and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2022.04.08.487606},
doi = {10.1101/2022.04.08.487606},
year = {2022},
date = {2022-04-08},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that scattered electrons carry informing about the charged state of atoms and can provide strong contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for drug discovery and electron microscopy can enable such visualization. Using subatomic resolution MicroED data obtained from triclinic hen egg-white lysozyme, we identified hundreds of individual hydrogen atom positions and directly visualize hydrogen bonding interactions and the charged states of residues. Over a third of all hydrogen atoms are identified from strong difference peaks, the most complete view of a macromolecular hydrogen network visualized by electron diffraction to date. These results show that MicroED can provide accurate structural information on hydrogen atoms and non-covalent hydrogen bonding interactions in macromolecules. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering and understanding the hydrogen bonding interactions underlying protein structure and function. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Clabbers, Max T B; Shiriaeva, Anna; Gonen, Tamir
MicroED: conception, practice and future opportunities Journal Article
In: IUCrJ, vol. 9, no. Pt 2, pp. 169–179, 2022, ISSN: 2052-2525.
@article{pmid35371502,
title = {MicroED: conception, practice and future opportunities},
author = {Max T B Clabbers and Anna Shiriaeva and Tamir Gonen},
doi = {10.1107/S2052252521013063},
issn = {2052-2525},
year = {2022},
date = {2022-03-01},
urldate = {2022-03-01},
journal = {IUCrJ},
volume = {9},
number = {Pt 2},
pages = {169--179},
abstract = {This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today's application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today's application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future.
Gallenito, Marc J; Gonen, Tamir
Studying membrane proteins with MicroED Journal Article
In: Biochem Soc Trans, vol. 50, no. 1, pp. 231–239, 2022, ISSN: 1470-8752.
@article{pmid35191473,
title = {Studying membrane proteins with MicroED},
author = {Marc J Gallenito and Tamir Gonen},
doi = {10.1042/BST20210911},
issn = {1470-8752},
year = {2022},
date = {2022-02-28},
urldate = {2022-02-01},
journal = {Biochem Soc Trans},
volume = {50},
number = {1},
pages = {231--239},
abstract = {The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED.
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, vol. 118, no. 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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, vol. 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.; Clabbers, Max T. B.; Hattne, Johan; Gonen, Tamir
Ab initio phasing macromolecular structures using electron-counted MicroED data Unpublished
bioRxiv, 2021.
@unpublished{Martynowycz2021,
title = {Ab initio phasing macromolecular structures using electron-counted MicroED data},
author = {Michael W. Martynowycz and Max T.B. Clabbers and Johan Hattne and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2021.10.16.464672},
doi = {10.1101/2021.10.16.464672},
year = {2021},
date = {2021-10-17},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that was collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion-beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous rotation data were collected using an ultra-low exposure rate on a Falcon 4 direct electron detector in electron-counting mode. For the first sample, triclinic lysozyme extending to 0.87 Å resolution, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a modest 1.5 Å resolution. These results demonstrate that macromolecules can be determined to sub-Ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data collected on a direct electron detector. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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, vol. 2, no. 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},
volume = {2},
number = {3},
pages = {100686},
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).},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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, vol. 118, no. 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},
pages = {e2106041118},
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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
Martynowycz, Michael W.; Clabbers, Max T. B.; Unge, Johan; Hattne, Johan; Gonen, Tamir
Benchmarking ideal sample thickness in cryo-EM using MicroED Unpublished
bioRxiv, 2021.
@unpublished{Martynowycz2021b,
title = {Benchmarking ideal sample thickness in cryo-EM using MicroED},
author = {Michael W. Martynowycz and Max T.B. Clabbers and Johan Unge and Johan Hattne and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2021.07.02.450941},
doi = {10.1101/2021.07.02.450941},
year = {2021},
date = {2021-07-03},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract The relationship between sample thickness and quality of data obtained by microcrystal electron diffraction (MicroED) is investigated. Several 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 scanning electron microscope (FIB/SEM) where the crystals were then systematically thinned into lamellae between 95 nm and 1650 nm thick. MicroED data were collected at either 120, 200, or 300 kV accelerating voltages. Lamellae thicknesses were converted to multiples of the calculated inelastic mean free path (MFP) of electrons at each accelerating voltage to allow the results to be compared on a common scale. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined from crystalline lamellae only 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 quality was insufficient to yield structures. No diffraction data were observed from lamellae thicker than four times the calculated inelastic mean free path. The quality of the determined structures and crystallographic statistics were similar for all lamellae up to 2x the inelastic mean free path in thickness, but quickly deteriorated at greater thicknesses. This study provides a benchmark with respect to the ideal limit for biological specimen thickness with implications for all cryo-EM methods. Significance A systematic investigation of the effects of thickness on electron scattering from protein crystals was previously not feasible, because there was no accurate method to control sample thickness. Here, the recently developed methods for preparing protein crystals into lamellae of precise thickness by ion-beam milling are used to investigate the effects of increasing sample thickness on MicroED data quality. These experiments were conducted using the three most common accelerating voltages in cryo-EM. Data across these accelerating voltages and thicknesses were compared on a common scale using their calculated inelastic mean free path lengths. It is found that structures may accurately be determined from crystals up to twice the inelastic mean free path length in thickness, regardless of the acceleration voltage.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Mu, Xuelang; Gillman, Cody; Nguyen, Chi; Gonen, Tamir
An Overview of Microcrystal Electron Diffraction (MicroED) Journal Article
In: Annu Rev Biochem, vol. 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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, vol. 29, no. 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.): vol. 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.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
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.): vol. 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.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
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, no. 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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, vol. 38, no. 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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., vol. 33, no. 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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, vol. 29, no. 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},
date = {2021-01-07},
urldate = {2021-01-07},
journal = {Structure},
volume = {29},
number = {1},
pages = {88--95.e2},
organization = {bioRxiv},
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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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, vol. 117, no. 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, vol. 28, no. 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.
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 Unpublished
bioRxiv, 2020.
@unpublished{Martynowycz2020,
title = {MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP},
author = {Michael W. Martynowycz and Anna Shiriaeva and Xuanrui Ge and Johan Hattne and Brent L. Nannenga and Vadim Cherezov and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2020.09.27.316109},
doi = {10.1101/2020.09.27.316109},
year = {2020},
date = {2020-09-28},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract G Protein-Coupled Receptors (GPCRs), or 7-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 signaling partners bound 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 cryoFIB milling. We determined the structure of the A2A receptor to 2.8 Å resolution and resolved an antagonist in its orthosteric ligand-binding site as well as 4 cholesterol molecules bound to the receptor. This study lays the groundwork for future GPCR structural studies using single microcrystals that would otherwise be impossible by other crystallographic methods. One sentence summary FIB milled LCP-GPCR structure determined by MicroED
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Nannenga, Brent L.; Gonen, Tamir
Microcrystal electron diffraction methodology and applications Journal Article
In: MRS Bulletin, vol. 44, pp. 956–960, 2020.
@article{Nannenga2020,
title = {Microcrystal electron diffraction methodology and applications},
author = {Brent L. Nannenga and Tamir Gonen},
doi = {10.1557/mrs.2019.287},
year = {2020},
date = {2020-09-27},
journal = {MRS Bulletin},
volume = {44},
pages = {956--960},
abstract = {Microcrystal electron diffraction (MicroED) is a cryo-electron microscopy technique that utilizes three-dimensional nano- and microcrystals for high-resolution structure determination. These extremely small crystals are several orders of magnitude smaller than what is used in conventional x-ray diffraction experiments. MicroED is capable of providing high-quality data from samples that would otherwise be considered useless for diffraction measurements. Since its initial implementation, MicroED has been used in the fields of structural biology, chemistry, and materials science. In this article, we provide an overview of the MicroED methodology as well as examples of how MicroED in cryo-electron microscopy has been used for structure determination of a variety of samples.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) is a cryo-electron microscopy technique that utilizes three-dimensional nano- and microcrystals for high-resolution structure determination. These extremely small crystals are several orders of magnitude smaller than what is used in conventional x-ray diffraction experiments. MicroED is capable of providing high-quality data from samples that would otherwise be considered useless for diffraction measurements. Since its initial implementation, MicroED has been used in the fields of structural biology, chemistry, and materials science. In this article, we provide an overview of the MicroED methodology as well as examples of how MicroED in cryo-electron microscopy has been used for structure determination of a variety of samples.
Martynowycz, Michael W.; Khan, Farha; Hattne, Johan; Abramson, Jeff; Gonen, Tamir
MicroED structure of lipid-embedded mammalian mitochondrial voltage dependent anion channel Unpublished
bioRxiv, 2020.
@unpublished{Martynowycz2020b,
title = {MicroED structure of lipid-embedded mammalian mitochondrial voltage dependent anion channel},
author = {Michael W. Martynowycz and Farha Khan and Johan Hattne and Jeff Abramson and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2020.09.17.302109},
doi = {10.1101/2020.09.17.302109},
year = {2020},
date = {2020-09-19},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract A near-atomic resolution structure of the mouse voltage dependent anion channel (mVDAC) is determined by combining cryogenic focused ion-beam (FIB) milling and microcrystal electron diffraction (MicroED). The crystals were grown in a viscous modified bicelle suspension which limited their size and made them unsuitable for conventional X-ray crystallography. Individual thin, plate-like crystals were identified using scanning electron microscopy (SEM) and focused ion-beam (FIB) imaging at high magnification. Three crystals were milled into thin lamellae. MicroED data were collected from each lamellae and merged to increase completeness. Unmodelled densities were observed between protein monomers, suggesting the presence of lipids that likely mediate crystal contacts. This work demonstrates the utility of milling membrane protein microcrystals grown in viscous media using a focused ion-beam for subsequent structure determination by MicroED for samples that are not otherwise tractable by other crystallographic methods. To our knowledge, the structure presented here is the first of a membrane protein crystallized in a lipid matrix and solved by MicroED. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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., vol. 76, no. 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., vol. 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 Unpublished
bioRxiv, 2020.
@unpublished{Martynowycz2020c,
title = {Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking},
author = {Michael W. Martynowycz and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2020.05.25.115246},
doi = {10.1101/2020.05.25.115246},
year = {2020},
date = {2020-05-26},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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, vol. 7, no. 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, vol. 28, no. 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.; Hattne, Johan; Gonen, Tamir
Experimental phasing of MicroED data using radiation damage Unpublished
bioRxiv, 2019.
@unpublished{Martynowycz2019,
title = {Experimental phasing of MicroED data using radiation damage},
author = {Michael W. Martynowycz and Johan Hattne and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/819086},
doi = {10.1101/819086},
year = {2019},
date = {2019-10-25},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract We present an example of an experimentally phased structure using only MicroED. The structure of a seven-residue peptide is determined 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 calculate a Patterson difference map and to identify a single heavy atom site, from which initial phases were generated. The structure was then completed by iterative cycles of modeling and refinement. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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, vol. 27, no. 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.
Wolff, Alexander M; Young, Iris D; Sierra, Raymond G; Brewster, Aaron S; Martynowycz, Michael W; Nango, Eriko; Sugahara, Michihiro; Nakane, Takanori; Ito, Kazutaka; Aquila, Andrew; Bhowmick, Asmit; Biel, Justin T; Carbajo, Sergio; Cohen, Aina E; Cortez, Saul; Gonzalez, Ana; Hino, Tomoya; Im, Dohyun; Koralek, Jake D; Kubo, Minoru; Lazarou, Tomas S; Nomura, Takashi; Owada, Shigeki; Samelson, Avi; Tanaka, Rie; Tanaka, Tomoyuki; Thompson, Erin M; van den Bedem, Henry; Woldeyes, Rahel A; Yumoto, Fumiaki; Zhao, Wei; Tono, Kensuke; Boutet, Sébastien; Iwata, So; Gonen, Tamir; Sauter, Nicholas K; Fraser, James S; Thompson, Michael C
bioRxiv, 2019.
@unpublished{Wolff2019,
title = {Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals},
author = {Alexander M Wolff and Iris D Young and Raymond G Sierra and Aaron S Brewster and Michael W Martynowycz and Eriko Nango and Michihiro Sugahara and Takanori Nakane and Kazutaka Ito and Andrew Aquila and Asmit Bhowmick and Justin T Biel and Sergio Carbajo and Aina E Cohen and Saul Cortez and Ana Gonzalez and Tomoya Hino and Dohyun Im and Jake D Koralek and Minoru Kubo and Tomas S Lazarou and Takashi Nomura and Shigeki Owada and Avi Samelson and Rie Tanaka and Tomoyuki Tanaka and Erin M Thompson and Henry van den Bedem and Rahel A Woldeyes and Fumiaki Yumoto and Wei Zhao and Kensuke Tono and Sébastien Boutet and So Iwata and Tamir Gonen and Nicholas K Sauter and James S Fraser and Michael C Thompson},
url = {http://biorxiv.org/lookup/doi/10.1101/767061},
doi = {10.1101/767061},
year = {2019},
date = {2019-09-12},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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 nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our 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 effect the crystal structure of the enzyme. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Hattne, Johan; Martynowycz, Michael W; Penczek, Pawel A; Gonen, Tamir
MicroED with the Falcon III direct electron detector Journal Article
In: IUCrJ, vol. 6, no. Pt 5, pp. 921–926, 2019.
@article{pmid31576224,
title = {MicroED with the Falcon III direct electron detector},
author = {Johan Hattne and Michael W Martynowycz and Pawel A Penczek and Tamir Gonen},
doi = {10.1107/S2052252519010583},
year = {2019},
date = {2019-09-01},
journal = {IUCrJ},
volume = {6},
number = {Pt 5},
pages = {921--926},
abstract = {Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.
Zhu, Lan; Bu, Guanhong; Jing, Liang; Shi, Dan; Gonen, Tamir; Liu, Wei; Nannenga, Brent L.
Structure determination from lipidic cubic phase embedded microcrystals by MicroED Unpublished
bioRxiv, 2019.
@unpublished{Zhu2019,
title = {Structure determination from lipidic cubic phase embedded microcrystals by MicroED},
author = {Lan Zhu and Guanhong Bu and Liang Jing and Dan Shi and Tamir Gonen and Wei Liu and Brent L. Nannenga},
url = {http://biorxiv.org/lookup/doi/10.1101/724575},
doi = {10.1101/724575},
year = {2019},
date = {2019-08-04},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. Because crystals grown in LCP can be limited in size, improved techniques for structure determination from these small crystals are important. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-TEM to collect electron diffraction data and determine high-resolution structures from very thin micro and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP. Proteinase K in LCP was used as a model system, and several LCP sample preparation strategies were tested. Among these, treatment with 2-Methyl-2,4-pentanediol (MPD) and lipase were both able to reduce the viscosity of the LCP and produce quality cryo-EM grids with well-diffracting crystals. These results set the stage for the use of MicroED to analyze other microcrystalline samples grown in LCP. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Warmack, Rebeccah A; Boyer, David R; Zee, Chih-Te; Richards, Logan S; Sawaya, Michael R; Cascio, Duilio; Gonen, Tamir; Eisenberg, David S; Clarke, Steven G
Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface Journal Article
In: Nat Commun, vol. 10, no. 1, pp. 3357, 2019.
@article{pmid31350392,
title = {Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface},
author = {Rebeccah A Warmack and David R Boyer and Chih-Te Zee and Logan S Richards and Michael R Sawaya and Duilio Cascio and Tamir Gonen and David S Eisenberg and Steven G Clarke},
doi = {10.1038/s41467-019-11183-z},
year = {2019},
date = {2019-07-26},
journal = {Nat Commun},
volume = {10},
number = {1},
pages = {3357},
abstract = {Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.
Ting, Chi P; Funk, Michael A; Halaby, Steve L; Zhang, Zhengan; Gonen, Tamir; van der Donk, Wilfred A
Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products Journal Article
In: Science, vol. 365, no. 6450, pp. 280–284, 2019.
@article{pmid31320540,
title = {Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products},
author = {Chi P Ting and Michael A Funk and Steve L Halaby and Zhengan Zhang and Tamir Gonen and Wilfred A van der Donk},
doi = {10.1126/science.aau6232},
year = {2019},
date = {2019-07-19},
journal = {Science},
volume = {365},
number = {6450},
pages = {280--284},
abstract = {Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA-dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid-derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid-derived natural products.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA-dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid-derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid-derived natural products.
Martynowycz, Michael W.; Gonen, Tamir
MicroED Structure of Hexagonal Ice Ih 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, vol. 201, pp. 77–80, 2019.
@article{pmid30986656,
title = {MicroED data collection with SerialEM},
author = {Jason M de la Cruz and Michael W Martynowycz and Johan Hattne and Tamir Gonen},
doi = {10.1016/j.ultramic.2019.03.009},
year = {2019},
date = {2019-06-01},
journal = {Ultramicroscopy},
volume = {201},
pages = {77--80},
abstract = {The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.