Investigator, Howard Hughes Medical Institute
Professor, Biological Chemistry and Physiology, 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 | MicroED Innovation Award |
| 2023 | A. L. Patterson award, ACA: The Structural Science Society |
Publications
2023
Bu, G; Danelius, E; Wieske, L. H; Gonen, T
Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED
bioRxiv, 2023.
@unpublished{Bu2023,
title = {Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED},
author = {G Bu and E Danelius and L.H Wieske and T Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2023.09.09.556999},
doi = {10.1101/2023.09.09.556999},
year = {2023},
date = {2023-09-10},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus infection. Its structures had been elusive to the public until recently when one of the crystal forms was solved by MicroED. In this work, we report the MicroED structures of two distinct polymorphic crystal forms of paritaprevir from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropylsulfonamide and methylpyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the NS3/4A serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acylsulfonamide inhibitors to the NS3/4A serine protease. In addition, this also demonstrate the opportunity of deriving different polymorphs and distinct macrocycle conformations from the same experiments using MicroED. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Lin, Jieye; Unge, Johan; Gonen, Tamir
Unraveling the Structure of Meclizine Dihydrochloride with MicroED
bioRxiv, 2023.
@unpublished{Lin2023b,
title = {Unraveling the Structure of Meclizine Dihydrochloride with MicroED},
author = {Jieye Lin and Johan Unge and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2023.09.05.556418},
doi = {10.1101/2023.09.05.556418},
year = {2023},
date = {2023-09-06},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract Meclizine (Antivert, Bonine) is a first-generation H1 antihistamine used in the treatment of motion sickness and vertigo. Despite its wide medical use for over 70 years, its crystal structure and the details of protein-drug interactions remained unknown. In this study, we used microcrystal electron diffraction (MicroED) to determine the three-dimensional (3D) crystal structure of meclizine dihydrochloride directly from a seemingly amorphous powder. Two racemic enantiomers (R/S) were found in the unit cell, which packed as repetitive double layers in the crystal lattice. The packing was made of multiple strong N-H···Cl- hydrogen bonding interactions and weak interactions like C-H···Cl- and pi-stacking. Molecular docking revealed the binding mechanism of meclizine to the histamine H1 receptor. A comparison of the docking complexes between histamine H1 receptor and meclizine or levocetirizine (a second-generation antihistamine) showed the conserved binding sites. This research illustrates the combined use of MicroED and molecular docking in unraveling protein-drug interactions for precision drug design and optimization. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Nannenga, Brent L.; Gonen, Tamir
The 2023 Structural Biology Summit at UCLA
In: Structure, vol. 31, pp. 1–2, 2023, ISSN: 0969-2126.
@article{Nannenga2023,
title = {The 2023 Structural Biology Summit at UCLA},
author = {Brent L. Nannenga and Tamir Gonen},
doi = {10.1016/j.str.2023.06.011},
issn = {0969-2126},
year = {2023},
date = {2023-08-16},
urldate = {2023-08-16},
journal = {Structure},
volume = {31},
pages = {1--2},
publisher = {Elsevier BV},
abstract = {In early 2023, the first Structural Biology Summit was held at the University of California, Los Angeles, which focused specifically on methods developments within the field of structural biology. This meeting report summarizes the 2023 Structural Biology summit and describes the main topics discussed during the meeting.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In early 2023, the first Structural Biology Summit was held at the University of California, Los Angeles, which focused specifically on methods developments within the field of structural biology. This meeting report summarizes the 2023 Structural Biology summit and describes the main topics discussed during the meeting.
Danelius, Emma; Bu, Guanhong; Wieske, Lianne; Gonen, Tamir
bioRxiv, 2023.
@unpublished{Danelius2023b,
title = {MicroED as a powerful tool for structure determination of macrocyclic drug compounds directly from their powder formulations},
author = {Emma Danelius and Guanhong Bu and Lianne Wieske and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2023.07.31.551405},
doi = {10.1101/2023.07.31.551405},
year = {2023},
date = {2023-08-01},
urldate = {2023-08-01},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles which have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material, and that different conformations of flexible compounds can be derived from the same experiment. These results will have impact on contemporary drug discovery as well as natural product exploration.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles which have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material, and that different conformations of flexible compounds can be derived from the same experiment. These results will have impact on contemporary drug discovery as well as natural product exploration.
Haymaker, Alison; Bardin, Andrey A.; Gonen, Tamir; Martynowycz, Michael W.; Nannenga, Brent L.
Structure determination of a DNA crystal by MicroED
In: Structure, 2023, ISSN: 0969-2126.
@article{Haymaker2023,
title = {Structure determination of a DNA crystal by MicroED},
author = {Alison Haymaker and Andrey A. Bardin and Tamir Gonen and Michael W. Martynowycz and Brent L. Nannenga},
doi = {10.1016/j.str.2023.07.005},
issn = {0969-2126},
year = {2023},
date = {2023-08-00},
journal = {Structure},
publisher = {Elsevier BV},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Shiriaeva, Anna; Martynowycz, Michael W; Nicolas, William J; Cherezov, Vadim; Gonen, Tamir
MicroED structure of the human vasopressin 1B receptor
bioRxiv, 2023.
@unpublished{pmid37461729,
title = {MicroED structure of the human vasopressin 1B receptor},
author = {Anna Shiriaeva and Michael W Martynowycz and William J Nicolas and Vadim Cherezov and Tamir Gonen},
doi = {10.1101/2023.07.05.547888},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {bioRxiv},
abstract = {The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 Å structure of the receptor in the crystallographic space group 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 Å structure of the receptor in the crystallographic space group 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods.
Gonen, Tamir
Reflections on two decades of MicroED development
In: RefleXions, no. 2, pp. 7–8, 2023.
@article{Gonen2023b,
title = {Reflections on two decades of MicroED development},
author = {Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/gonen-reflexions-2023.pdf, Main text},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {RefleXions},
number = {2},
pages = {7--8},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Gillman, Cody; Nicolas, William J; Martynowycz, Michael W; Gonen, Tamir
Design and implementation of suspended drop crystallization
In: IUCrJ, vol. 10, iss. 4, 2023, ISSN: 2052-2525.
@article{pmid37223996,
title = {Design and implementation of suspended drop crystallization},
author = {Cody Gillman and William J Nicolas and Michael W Martynowycz and Tamir Gonen},
doi = {10.1107/S2052252523004141},
issn = {2052-2525},
year = {2023},
date = {2023-07-00},
urldate = {2023-07-01},
journal = {IUCrJ},
volume = {10},
issue = {4},
abstract = {In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.
Hattne, Johan; Clabbers, Max T. B.; Martynowycz, Michael W.; Gonen, Tamir
bioRxiv, 2023.
@unpublished{Hattne2023,
title = {Electron-counting in MicroED},
author = {Johan Hattne and Max T. B. Clabbers and Michael W. Martynowycz and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2023.06.29.547123},
doi = {10.1101/2023.06.29.547123},
year = {2023},
date = {2023-06-30},
urldate = {2023-06-30},
publisher = {Cold Spring Harbor Laboratory},
abstract = {The combination of high sensitivity and rapid readout makes it possible for electron-counting detectors to record cryogenic electron microscopy data faster and more accurately without increasing the exposure. This is especially useful for MicroED of macromolecular crystals where the strength of the diffracted signal at high resolution is comparable to the surrounding background. The ability to decrease the exposure also alleviates concerns about radiation damage which limits the information that can be recovered from a diffraction measurement. However, the dynamic range of electron-counting detectors requires careful data collection to avoid errors from coincidence loss. Nevertheless, these detectors are increasingly deployed in cryo-EM facilities, and several have been successfully used for MicroED. Provided coincidence loss can be minimized, electron-counting detectors bring high potential rewards.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
The combination of high sensitivity and rapid readout makes it possible for electron-counting detectors to record cryogenic electron microscopy data faster and more accurately without increasing the exposure. This is especially useful for MicroED of macromolecular crystals where the strength of the diffracted signal at high resolution is comparable to the surrounding background. The ability to decrease the exposure also alleviates concerns about radiation damage which limits the information that can be recovered from a diffraction measurement. However, the dynamic range of electron-counting detectors requires careful data collection to avoid errors from coincidence loss. Nevertheless, these detectors are increasingly deployed in cryo-EM facilities, and several have been successfully used for MicroED. Provided coincidence loss can be minimized, electron-counting detectors bring high potential rewards.
Lin, Jieye; Unge, Johan; Gonen, Tamir
Distinct Conformations of Mirabegron Determined by MicroED
bioRxiv, 2023.
@unpublished{Lin2023,
title = {Distinct Conformations of Mirabegron Determined by MicroED},
author = {Jieye Lin and Johan Unge and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2023.06.28.546957},
doi = {10.1101/2023.06.28.546957},
year = {2023},
date = {2023-06-28},
urldate = {2023-06-28},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Mirabegron, commonly known as “Myrbetriq”, has been widely prescribed as a medicine for overactive bladder syndrome for over a decade. However, the structure of the drug and what conformational changes it may undergo upon binding its receptor remain unknown. In this study, we employed microcrystal electron diffraction (MicroED) to reveal its elusive three-dimensional (3D) structure. We find that the drug adopts two distinct conformational states (conformers) within the asymmetric unit. Analysis of hydrogen bonding and packing demonstrated that the hydrophilic groups were embedded within the crystal lattice, resulting in a hydrophobic surface and low water solubility. Structural comparison revealed the presence of trans- and cis-forms in conformers 1 and 2, respectively. Comparison of the structures of Mirabegron alone with that of the drug bound to its receptor, 1 the beta 3 adrenergic receptor (β3AR) suggests that the drug undergoes major conformational change to fit in the receptor agonist binding site. This research highlights the efficacy of MicroED in determining the unknown and polymorphic structures of active pharmaceutical ingredients (APIs) directly from powders.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Mirabegron, commonly known as “Myrbetriq”, has been widely prescribed as a medicine for overactive bladder syndrome for over a decade. However, the structure of the drug and what conformational changes it may undergo upon binding its receptor remain unknown. In this study, we employed microcrystal electron diffraction (MicroED) to reveal its elusive three-dimensional (3D) structure. We find that the drug adopts two distinct conformational states (conformers) within the asymmetric unit. Analysis of hydrogen bonding and packing demonstrated that the hydrophilic groups were embedded within the crystal lattice, resulting in a hydrophobic surface and low water solubility. Structural comparison revealed the presence of trans- and cis-forms in conformers 1 and 2, respectively. Comparison of the structures of Mirabegron alone with that of the drug bound to its receptor, 1 the beta 3 adrenergic receptor (β3AR) suggests that the drug undergoes major conformational change to fit in the receptor agonist binding site. This research highlights the efficacy of MicroED in determining the unknown and polymorphic structures of active pharmaceutical ingredients (APIs) directly from powders.
Unge, Johan; Lin, Jieye; Weaver, Sara J; Her, Ampon Sae; Gonen, Tamir
Autonomous MicroED data collection enables compositional analysis
ChemRxiv, 2023.
@unpublished{Unge2023,
title = {Autonomous MicroED data collection enables compositional analysis},
author = {Johan Unge and Jieye Lin and Sara J Weaver and Ampon Sae Her and Tamir Gonen},
url = {https://chemrxiv.org/engage/chemrxiv/article-details/6465a5f8fb40f6b3eec2aaa7},
doi = {10.26434/chemrxiv-2023-8qvwg},
year = {2023},
date = {2023-05-18},
urldate = {2023-05-18},
publisher = {American Chemical Society (ACS)},
abstract = {MicroED is an effective method for analyzing the structural properties of sub-micron crystals, which are frequently found in small-molecule powders. By developing and using an autonomous and high throughput approach to MicroED, we demonstrate the expansion of capabilities and the possibility of performing complete compositional analysis of complex samples. With the use of SerialEM for data collection of thousands of datasets from thousands of crystals and an automated processing pipeline, compositional analysis of complex mixtures of organic and inorganic compounds can be accurately executed. Quantitative analysis suitable for compounds having similar chemical properties can be made on the fly. These compounds can be distinguished by their crystal structure properties prior to structure solution. Additionally, with sufficient statistics from the autonomous approach, even small amounts of compounds in mixtures can be reliably detected. Finally, atomic structures can be determined from the thousands of data sets.},
howpublished = {ChemRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
MicroED is an effective method for analyzing the structural properties of sub-micron crystals, which are frequently found in small-molecule powders. By developing and using an autonomous and high throughput approach to MicroED, we demonstrate the expansion of capabilities and the possibility of performing complete compositional analysis of complex samples. With the use of SerialEM for data collection of thousands of datasets from thousands of crystals and an automated processing pipeline, compositional analysis of complex mixtures of organic and inorganic compounds can be accurately executed. Quantitative analysis suitable for compounds having similar chemical properties can be made on the fly. These compounds can be distinguished by their crystal structure properties prior to structure solution. Additionally, with sufficient statistics from the autonomous approach, even small amounts of compounds in mixtures can be reliably detected. Finally, atomic structures can be determined from the thousands of data sets.
Nguyen, Chi; Lei, Hsiang-Ting; Lai, Louis Tung Faat; Gallenito, Marc J; Mu, Xuelang; Matthies, Doreen; Gonen, Tamir
Lipid flipping in the omega-3 fatty-acid transporter
In: Nat Commun, vol. 14, no. 1, pp. 2571, 2023, ISSN: 2041-1723.
@article{pmid37156797,
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 Xuelang Mu and Doreen Matthies and Tamir Gonen},
doi = {10.1038/s41467-023-37702-7},
issn = {2041-1723},
year = {2023},
date = {2023-05-01},
urldate = {2023-05-01},
journal = {Nat Commun},
volume = {14},
number = {1},
pages = {2571},
abstract = {Mfsd2a is the transporter for docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral and motor dysfunctions to microcephaly. Mfsd2a transports long-chain unsaturated fatty-acids, including DHA and α-linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with the 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 remains unclear. Here, we report five single-particle cryo-EM structures of Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and displaying lipid-like densities modeled as ALA-LPC at four distinct positions. These Mfsd2a snapshots detail the flipping mechanism for lipid-LPC from outer to inner membrane leaflet and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mfsd2a is the transporter for docosahexaenoic acid (DHA), an omega-3 fatty acid, across the blood brain barrier (BBB). Defects in Mfsd2a are linked to ailments from behavioral and motor dysfunctions to microcephaly. Mfsd2a transports long-chain unsaturated fatty-acids, including DHA and α-linolenic acid (ALA), that are attached to the zwitterionic lysophosphatidylcholine (LPC) headgroup. Even with the 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 remains unclear. Here, we report five single-particle cryo-EM structures of Danio rerio Mfsd2a (drMfsd2a): in the inward-open conformation in the ligand-free state and displaying lipid-like densities modeled as ALA-LPC at four distinct positions. These Mfsd2a snapshots detail the flipping mechanism for lipid-LPC from outer to inner membrane leaflet and release for membrane integration on the cytoplasmic side. These results also map Mfsd2a mutants that disrupt lipid-LPC transport and are associated with disease.
Gillman, Cody; Patel, Khushboo; Unge, Johan; Gonen, Tamir
The structure of the neurotoxin palytoxin determined by MicroED
bioRxiv, 2023.
@unpublished{Gillman2023,
title = {The structure of the neurotoxin palytoxin determined by MicroED},
author = {Cody Gillman and Khushboo Patel and Johan Unge and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2023.03.31.535166},
doi = {10.1101/2023.03.31.535166},
year = {2023},
date = {2023-04-02},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract Palytoxin (PTX) is a potent neurotoxin found in marine animals that can cause serious symptoms such as muscle contractions, haemolysis of red blood cells and potassium leakage. Despite years of research, very little is known about the mechanism of PTX. However, recent advances in the field of cryoEM, specifically the use of microcrystal electron diffraction (MicroED), have allowed us to determine the structure of PTX. It was discovered that PTX folds into a hairpin motif and is able to bind to the extracellular gate of Na,K-ATPase, which is responsible for maintaining the electrochemical gradient across the plasma membrane. These findings, along with molecular docking simulations, have provided important insights into the mechanism of PTX and can potentially aid in the development of molecular agents for treating cases of PTX exposure. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Haymaker, Alison; Bardin, Andrey A; Gonen, Tamir; Martynowycz, Michael W; Nannenga, Brent L
Structure determination of a DNA crystal by MicroED
bioRxiv, 2023.
@unpublished{pmid37163108,
title = {Structure determination of a DNA crystal by MicroED},
author = {Alison Haymaker and Andrey A Bardin and Tamir Gonen and Michael W Martynowycz and Brent L Nannenga},
doi = {10.1101/2023.04.25.538338},
year = {2023},
date = {2023-04-01},
urldate = {2023-04-01},
journal = {bioRxiv},
abstract = {Microcrystal electron diffraction (MicroED) is a powerful tool for determining high-resolution structures of microcrystals from a diverse array of biomolecular, chemical, and material samples. In this study, we apply MicroED to DNA crystals, which have not been previously analyzed using this technique. We utilized the d(CGCGCG) DNA duplex as a model sample and employed cryo-FIB milling to create thin lamella for diffraction data collection. The MicroED data collection and subsequent processing resulted in a 1.10 Å resolution structure of the d(CGCGCG) DNA, demonstrating the successful application of cryo-FIB milling and MicroED to the investigation of nucleic acid crystals.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Microcrystal electron diffraction (MicroED) is a powerful tool for determining high-resolution structures of microcrystals from a diverse array of biomolecular, chemical, and material samples. In this study, we apply MicroED to DNA crystals, which have not been previously analyzed using this technique. We utilized the d(CGCGCG) DNA duplex as a model sample and employed cryo-FIB milling to create thin lamella for diffraction data collection. The MicroED data collection and subsequent processing resulted in a 1.10 Å resolution structure of the d(CGCGCG) DNA, demonstrating the successful application of cryo-FIB milling and MicroED to the investigation of nucleic acid crystals.
Gillman, Cody; Martynowycz, Michael; Nicolas, William Jules; Gonen, Tamir
Design and implementation of suspended drop crystallization
bioRxiv, 2023.
@unpublished{Gonen2023,
title = {Design and implementation of suspended drop crystallization},
author = {Cody Gillman and Michael Martynowycz and William Jules Nicolas and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2023.03.28.534639},
doi = {10.1101/2023.03.28.534639},
year = {2023},
date = {2023-03-28},
urldate = {2023-03-28},
publisher = {Cold Spring Harbor Laboratory},
abstract = {We have developed a novel crystal growth method known as suspended drop crystallization. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber which we designed, allowing for vapor diffusion to occur from both sides of the drop. A UV transparent window above and below the grid enables the monitoring of crystal growth via light, UV, or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for x-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, we grew crystals of the enzyme proteinase K and determined its structure by MicroED following FIB/SEM milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress, and/or suffering from preferred orientation on EM grids. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
<jats:p>We have developed a novel crystal growth method known as suspended drop crystallization. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber which we designed, allowing for vapor diffusion to occur from both sides of the drop. A UV transparent window above and below the grid enables the monitoring of crystal growth via light, UV, or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for x-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, we grew crystals of the enzyme proteinase K and determined its structure by MicroED following FIB/SEM milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress, and/or suffering from preferred orientation on EM grids.</jats:p>
Danelius, Emma; Porter, Nicholas J.; Unge, Johan; Arnold, Frances H.; Gonen, Tamir
MicroED Structure of a Protoglobin Reactive Carbene Intermediate
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
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
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
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
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},
urldate = {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 using ab 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 an Aeropyrum pernixprotoglobin (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 of Ape 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 engineered Ape 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}
}
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 using ab 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 an Aeropyrum pernixprotoglobin (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 of Ape 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 engineered Ape 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.
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
In: J Struct Biol, vol. 214, iss. 4, 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},
volume = {214},
issue = {4},
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
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}
}
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
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.; Gonen, Tamir
Unlocking the potential of microcrystal electron diffraction
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 = {2022-06-00},
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.
Martynowycz, Michael W; Clabbers, Max T B; Hattne, Johan; Gonen, Tamir
Ab initio phasing macromolecular structures using electron-counted MicroED data
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; Danelius, Emma; Gonen, Tamir; Arnold, Frances
Biocatalytic Carbene Transfer Using Diazirines
ChemRxiv, 2022.
@unpublished{Porter2022,
title = {Biocatalytic Carbene Transfer Using Diazirines},
author = {Nicholas Porter and Emma Danelius and Tamir Gonen and Frances Arnold},
doi = {10.26434/chemrxiv-2022-9v4j1},
year = {2022},
date = {2022-05-04},
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 Aeropyrum pernix protoglobin (ApePgb) that use diazirines as carbene precursors. While the enhanced stability of diazir- ines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered ApePgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an ApePgb 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.},
howpublished = {ChemRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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 Aeropyrum pernix protoglobin (ApePgb) that use diazirines as carbene precursors. While the enhanced stability of diazir- ines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered ApePgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an ApePgb 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.
Porter, Nicholas J; Danelius, Emma; Gonen, Tamir; Arnold, Frances H
Biocatalytic Carbene Transfer Using Diazirines
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
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
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
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
Gonen, Tamir; Nannenga, Brent (Ed.)
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 = {2021-12-21},
urldate = {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.
Martynowycz, Michael W; Clabbers, Max T B; Unge, Johan; Hattne, Johan; Gonen, Tamir
Benchmarking the ideal sample thickness in cryo-EM
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
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
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
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
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
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)
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
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
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
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
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.
Halaby, Steve; Martynowycz, Michael W.; Zhu, Ziyue; Tretiak, Sergei; Zhugayevych, Andriy; Gonen, Tamir; and Martin Seifrid,
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
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.
2020
Martynowycz, M W; Khan, F; Hattne, J; Abramson, J; Gonen, T
MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel
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
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
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
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
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}
}