Publications
Referencing us
MEDIC must be acknowledged in all publications that resulted from either collaboration with the Center or with individual personnel of the Center or utilized services of the Center. The NIH grant funding the MEDIC project is NIH/GM P41 136508.
As in all scientific publications, co-authorship for a member of the Scientific Staff is warranted when an individual has made a significant contribution to the scientific work being described.
Data policy
All data collected for structural purposes will be made publicly available upon publication. Structures will be deposited in the protein structure data bank (PDB) and the electron microscopy data bank (EMDB) prior to publication. All primary data associated with a structure will be made available to the community upon request.
Sharing of materials
All materials, plasmids, peptides, and protein fragments will be made available to the research community in accordance with NIH guidelines and in coordination with the UCLA technology development group (TDG) governing material transfer agreements (MTA).
Sharing of software and algorithms
All software and programs used for analysis or structure determination will be made available either as pseudocode or as standalone scripts/programs in accordance with NIH guidelines and in coordination with the UCLA technology development group (TDG) governing intellectual property dissemination and materials transfer agreements (MTA).
Publication list
Click the Altmetric or PlumX links to show article-level metrics. The MEDIC H-index = 6.
2023
Danelius, Emma; Porter, Nicholas J; Unge, Johan; Arnold, Frances H; Gonen, Tamir
MicroED Structure of a Protoglobin Reactive Carbene Intermediate Journal Article
In: J Am Chem Soc, vol. 145, no. 13, pp. 7159–7165, 2023, ISSN: 1520-5126.
@article{pmid36948184,
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 = {2023-04-01},
urldate = {2023-04-01},
journal = {J Am Chem Soc},
volume = {145},
number = {13},
pages = {7159--7165},
abstract = {Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using methods. However, only a couple of novel protein structures have thus far been derived by MicroED. 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 protoglobin (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 α helix of Pgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered 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 the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using methods. However, only a couple of novel protein structures have thus far been derived by MicroED. 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 protoglobin (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 α helix of Pgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered 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 the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.
Danelius, Emma; Patel, Khushboo; Gonzalez, Brenda; Gonen, Tamir
MicroED in drug discovery Journal Article
In: Curr Opin Struct Biol, vol. 79, pp. 102549, 2023, ISSN: 1879-033X.
@article{pmid36821888,
title = {MicroED in drug discovery},
author = {Emma Danelius and Khushboo Patel and Brenda Gonzalez and Tamir Gonen},
doi = {10.1016/j.sbi.2023.102549},
issn = {1879-033X},
year = {2023},
date = {2023-04-01},
urldate = {2023-04-01},
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.
Gillman, Cody; Patel, Khushboo; Unge, Johan; Gonen, Tamir
The structure of the neurotoxin palytoxin determined by MicroED Bachelor Thesis
2023.
@bachelorthesis{pmid37034718,
title = {The structure of the neurotoxin palytoxin determined by MicroED},
author = {Cody Gillman and Khushboo Patel and Johan Unge and Tamir Gonen},
doi = {10.1101/2023.03.31.535166},
year = {2023},
date = {2023-04-01},
urldate = {2023-04-01},
journal = {bioRxiv},
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.},
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pubstate = {published},
tppubtype = {bachelorthesis}
}
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.
Gillman, Cody; Nicolas, William J; Martynowycz, Michael W; Gonen, Tamir
Design and implementation of suspended drop crystallization Bachelor Thesis
2023.
@bachelorthesis{pmid37034794,
title = {Design and implementation of suspended drop crystallization},
author = {Cody Gillman and William J Nicolas and Michael W Martynowycz and Tamir Gonen},
doi = {10.1101/2023.03.28.534639},
year = {2023},
date = {2023-03-01},
urldate = {2023-03-01},
journal = {bioRxiv},
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.},
keywords = {},
pubstate = {published},
tppubtype = {bachelorthesis}
}
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.
Martynowycz, Michael W; Shiriaeva, Anna; Clabbers, Max T B; Nicolas, William J; Weaver, Sara J; Hattne, Johan; Gonen, Tamir
A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals Journal Article
In: Nat Commun, vol. 14, 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 = {2023-02-01},
urldate = {2023-02-01},
journal = {Nat Commun},
volume = {14},
number = {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.
2022
Clabbers, Max T B; Martynowycz, Michael W; Hattne, Johan; Nannenga, Brent L; Gonen, Tamir
Electron-counting MicroED data with the K2 and K3 direct electron detectors Journal Article
In: J Struct Biol, vol. 214, no. 4, pp. 107886, 2022, ISSN: 1095-8657.
@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},
issn = {1095-8657},
year = {2022},
date = {2022-12-01},
urldate = {2022-12-01},
journal = {J Struct Biol},
volume = {214},
number = {4},
pages = {107886},
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 resolution. Even though a beam stop was not used with the K3 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 resolution. Even though a beam stop was not used with the K3 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.
Clabbers, Max T. B.; Martynowycz, Michael W.; Hattne, Johan; Gonen, Tamir
Hydrogens and hydrogen-bond networks in macromolecular MicroED data Journal Article
In: J Struct Biol X, vol. 6, pp. 100078, 2022.
@article{nokey,
title = {Hydrogens and hydrogen-bond networks in macromolecular MicroED data},
author = {Max T.B. Clabbers and Michael W. Martynowycz and Johan Hattne and Tamir Gonen},
doi = {10.1016/j.yjsbx.2022.100078},
year = {2022},
date = {2022-11-10},
urldate = {2022-11-10},
journal = {J Struct Biol X},
volume = {6},
pages = {100078},
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.
Martynowycz, Michael W.; Shiriaeva, Anna; Clabbers, Max T. B.; Nicolas, William J.; Weaver, Sara J.; Hattne, Johan; Gonen, Tamir
2022, visited: 26.07.2022.
@online{nokey,
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},
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.
Martynowycz, Michael W; Clabbers, Max T B; Hattne, Johan; Gonen, Tamir
Ab initio phasing macromolecular structures using electron-counted MicroED data Journal Article
In: Nat Methods, vol. 19, no. 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 = {2022-06-01},
urldate = {2022-06-01},
journal = {Nat Methods},
volume = {19},
number = {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.
Martynowycz, Mike; Gonen, Tamir
Unlocking the potential of MICROCRYSTAL ELECTRON DIFFRACTION Journal Article
In: Phys Today, vol. 75, no. 6, pp. 38–42, 2022, ISSN: 0031-9228.
@article{pmid36969383,
title = {Unlocking the potential of MICROCRYSTAL ELECTRON DIFFRACTION},
author = {Mike Martynowycz and Tamir Gonen},
doi = {10.1063/pt.3.5019},
issn = {0031-9228},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {Phys Today},
volume = {75},
number = {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.
2021
Martynowycz, Michael W; Gonen, Tamir
Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED) Journal Article
In: STAR Protoc, vol. 2, no. 3, pp. 100686, 2021, ISSN: 2666-1667.
@article{pmid34382014,
title = {Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED)},
author = {Michael W Martynowycz and Tamir Gonen},
doi = {10.1016/j.xpro.2021.100686},
issn = {2666-1667},
year = {2021},
date = {2021-09-01},
urldate = {2021-09-01},
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).
Danelius, Emma; Gonen, Tamir
Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED Journal Article
In: Methods Mol Biol, vol. 2305, pp. 323–342, 2021, ISSN: 1940-6029.
@article{pmid33950397,
title = {Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED},
author = {Emma Danelius and Tamir Gonen},
doi = {10.1007/978-1-0716-1406-8_16},
issn = {1940-6029},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2305},
pages = {323--342},
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 = {article}
}
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, Michael W; Gonen, Tamir
Studying Membrane Protein Structures by MicroED Journal Article
In: Methods Mol Biol, vol. 2302, pp. 137–151, 2021, ISSN: 1940-6029.
@article{pmid33877626,
title = {Studying Membrane Protein Structures by MicroED},
author = {Michael W Martynowycz and Tamir Gonen},
doi = {10.1007/978-1-0716-1394-8_8},
issn = {1940-6029},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2302},
pages = {137--151},
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 = {article}
}
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.
Mu, Xuelang; Gillman, Cody; Nguyen, Chi; Gonen, Tamir
An Overview of Microcrystal Electron Diffraction (MicroED) Journal Article
In: Annu Rev Biochem, vol. 90, pp. 431–450, 2021, ISSN: 1545-4509.
@article{pmid34153215,
title = {An Overview of Microcrystal Electron Diffraction (MicroED)},
author = {Xuelang Mu and Cody Gillman and Chi Nguyen and Tamir Gonen},
doi = {10.1146/annurev-biochem-081720-020121},
issn = {1545-4509},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
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.
Martynowycz, Michael W; Gonen, Tamir
Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking Journal Article
In: Structure, vol. 29, no. 1, pp. 88–95.e2, 2021, ISSN: 1878-4186.
@article{pmid33007196,
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},
issn = {1878-4186},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Structure},
volume = {29},
number = {1},
pages = {88--95.e2},
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.
Martynowycz, Michael W; Gonen, Tamir
Microcrystal Electron Diffraction of Small Molecules Journal Article
In: J Vis Exp, no. 169, 2021, ISSN: 1940-087X.
@article{pmid33779618,
title = {Microcrystal Electron Diffraction of Small Molecules},
author = {Michael W Martynowycz and Tamir Gonen},
doi = {10.3791/62313},
issn = {1940-087X},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {J Vis Exp},
number = {169},
abstract = {A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.
Danelius, Emma; Halaby, Steve; van der Donk, Wilfred A; Gonen, Tamir
MicroED in natural product and small molecule research Journal Article
In: Nat Prod Rep, vol. 38, no. 3, pp. 423–431, 2021, ISSN: 1460-4752.
@article{pmid32939523,
title = {MicroED in natural product and small molecule research},
author = {Emma Danelius and Steve Halaby and Wilfred A van der Donk and Tamir Gonen},
doi = {10.1039/d0np00035c},
issn = {1460-4752},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Nat Prod Rep},
volume = {38},
number = {3},
pages = {423--431},
abstract = {Covering: 2013 to 2020The electron cryo-microscopy (cryo-EM) method Microcrystal Electron Diffraction (MicroED) allows the collection of high-resolution structural data from vanishingly small crystals that appear like amorphous powders or very fine needles. Since its debut in 2013, data collection and analysis schemes have been fine-tuned, and there are currently close to 100 structures determined by MicroED. Although originally developed to study proteins, MicroED is also very powerful for smaller systems, with some recent and very promising examples from the field of natural products. Herein, we review what has been achieved so far and provide examples of natural product structures, as well as demonstrate the expected future impact of MicroED to the field of natural product and small molecule research.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Covering: 2013 to 2020The electron cryo-microscopy (cryo-EM) method Microcrystal Electron Diffraction (MicroED) allows the collection of high-resolution structural data from vanishingly small crystals that appear like amorphous powders or very fine needles. Since its debut in 2013, data collection and analysis schemes have been fine-tuned, and there are currently close to 100 structures determined by MicroED. Although originally developed to study proteins, MicroED is also very powerful for smaller systems, with some recent and very promising examples from the field of natural products. Herein, we review what has been achieved so far and provide examples of natural product structures, as well as demonstrate the expected future impact of MicroED to the field of natural product and small molecule research.
Martynowycz, Michael W; Shiriaeva, Anna; Ge, Xuanrui; Hattne, Johan; Nannenga, Brent L; Cherezov, Vadim; Gonen, Tamir
MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP Journal Article
In: Proc Natl Acad Sci U S A, vol. 118, no. 36, 2021, ISSN: 1091-6490.
@article{pmid34462357,
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},
doi = {10.1073/pnas.2106041118},
issn = {1091-6490},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {118},
number = {36},
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 A 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 A 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 A 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 A 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 the ideal sample thickness in cryo-EM Journal Article
In: Proc Natl Acad Sci U S A, vol. 118, no. 49, 2021, ISSN: 1091-6490.
@article{pmid34873060,
title = {Benchmarking the ideal sample thickness in cryo-EM},
author = {Michael W Martynowycz and Max T B Clabbers and Johan Unge and Johan Hattne and Tamir Gonen},
doi = {10.1073/pnas.2108884118},
issn = {1091-6490},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
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.
Lei, Hsiang-Ting; Mu, Xuelang; Hattne, Johan; Gonen, Tamir
A conformational change in the N terminus of SLC38A9 signals mTORC1 activation Journal Article
In: Structure, vol. 29, no. 5, pp. 426–432.e8, 2021, ISSN: 1878-4186.
@article{pmid33296665,
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},
issn = {1878-4186},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
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.
2020
Richards, Logan S; Millán, Claudia; Miao, Jennifer; Martynowycz, Michael W; Sawaya, Michael R; Gonen, Tamir; Borges, Rafael J; Usón, Isabel; Rodriguez, Jose A
Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER Journal Article
In: Acta Crystallogr D Struct Biol, vol. 76, no. Pt 8, pp. 703–712, 2020, ISSN: 2059-7983.
@article{pmid32744252,
title = {Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER},
author = {Logan S Richards and Claudia Millán and Jennifer Miao and Michael W Martynowycz and Michael R Sawaya and Tamir Gonen and Rafael J Borges and Isabel Usón and Jose A Rodriguez},
doi = {10.1107/S2059798320008049},
issn = {2059-7983},
year = {2020},
date = {2020-08-01},
urldate = {2020-08-01},
journal = {Acta Crystallogr D Struct Biol},
volume = {76},
number = {Pt 8},
pages = {703--712},
abstract = {Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose.
Nguyen, Chi; Gonen, Tamir
Beyond protein structure determination with MicroED Journal Article
In: Curr Opin Struct Biol, vol. 64, pp. 51–58, 2020, ISSN: 1879-033X.
@article{pmid32610218,
title = {Beyond protein structure determination with MicroED},
author = {Chi Nguyen and Tamir Gonen},
doi = {10.1016/j.sbi.2020.05.018},
issn = {1879-033X},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Curr Opin Struct Biol},
volume = {64},
pages = {51--58},
abstract = {Microcrystal electron diffraction (MicroED) was first coined and developed in 2013 at the Janelia Research Campus as a new modality in electron cryomicroscopy (cryoEM). Since then, MicroED has not only made important contributions in pushing the resolution limits of cryoEM protein structure characterization but also of peptides, small-organic and inorganic molecules, and natural-products that have resisted structure determination by other methods. This review showcases important recent developments in MicroED, highlighting the importance of the technique in fields of studies beyond protein structure determination where MicroED is beginning to have paradigm shifting roles.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) was first coined and developed in 2013 at the Janelia Research Campus as a new modality in electron cryomicroscopy (cryoEM). Since then, MicroED has not only made important contributions in pushing the resolution limits of cryoEM protein structure characterization but also of peptides, small-organic and inorganic molecules, and natural-products that have resisted structure determination by other methods. This review showcases important recent developments in MicroED, highlighting the importance of the technique in fields of studies beyond protein structure determination where MicroED is beginning to have paradigm shifting roles.
Martynowycz, Michael W; Hattne, Johan; Gonen, Tamir
Experimental Phasing of MicroED Data Using Radiation Damage Journal Article
In: Structure, vol. 28, no. 4, pp. 458–464.e2, 2020, ISSN: 1878-4186.
@article{pmid32023481,
title = {Experimental Phasing of MicroED Data Using Radiation Damage},
author = {Michael W Martynowycz and Johan Hattne and Tamir Gonen},
doi = {10.1016/j.str.2020.01.008},
issn = {1878-4186},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Structure},
volume = {28},
number = {4},
pages = {458--464.e2},
abstract = {We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present an example of an experimentally phased structure using only MicroED data. The structure of a seven-residue peptide is solved starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous-rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a "low-dose dataset." The data from the second pass were similarly merged to form a "damaged dataset." Differences between these two datasets were used to identify a single heavy-atom site from a Patterson difference map, and initial phases were generated. Finally, the structure was completed by iterative cycles of modeling and refinement.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present an example of an experimentally phased structure using only MicroED data. The structure of a seven-residue peptide is solved starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous-rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a "low-dose dataset." The data from the second pass were similarly merged to form a "damaged dataset." Differences between these two datasets were used to identify a single heavy-atom site from a Patterson difference map, and initial phases were generated. Finally, the structure was completed by iterative cycles of modeling and refinement.
Martynowycz, Michael W; Khan, Farha; Hattne, Johan; Abramson, Jeff; Gonen, Tamir
MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel Journal Article
In: Proc Natl Acad Sci U S A, vol. 117, no. 51, pp. 32380–32385, 2020, ISSN: 1091-6490.
@article{pmid33293416,
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},
doi = {10.1073/pnas.2020010117},
issn = {1091-6490},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {117},
number = {51},
pages = {32380--32385},
abstract = {A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.
Zhu, Lan; Bu, Guanhong; Jing, Liang; Shi, Dan; Lee, Ming-Yue; Gonen, Tamir; Liu, Wei; Nannenga, Brent L
Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED Journal Article
In: Structure, vol. 28, no. 10, pp. 1149–1159.e4, 2020, ISSN: 1878-4186.
@article{pmid32735770,
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},
doi = {10.1016/j.str.2020.07.006},
issn = {1878-4186},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
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 A 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 A 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.