2024
204.
Nicolas, William J.; Gillman, Cody; Weaver, Sara J.; Clabbers, Max T. B.; Shiriaeva, Anna; Her, Ampon Sae; Martynowycz, Michael W.; Gonen, Tamir
Comprehensive microcrystal electron diffraction sample preparation for cryo-EM
In: Nat Protoc, 2024, ISSN: 1750-2799.
@article{Nicolas2024b,
title = {Comprehensive microcrystal electron diffraction sample preparation for cryo-EM},
author = {William J. Nicolas and Cody Gillman and Sara J. Weaver and Max T. B. Clabbers and Anna Shiriaeva and Ampon Sae Her and Michael W. Martynowycz and Tamir Gonen},
doi = {10.1038/s41596-024-01088-7},
issn = {1750-2799},
year = {2024},
date = {2024-12-20},
urldate = {2024-12-20},
journal = {Nat Protoc},
publisher = {Springer Science and Business Media LLC},
abstract = {Microcrystal electron diffraction (MicroED) has advanced structural methods across a range of sample types, from small molecules to proteins. This cryogenic electron microscopy (cryo-EM) technique involves the continuous rotation of small 3D crystals in the electron beam, while a high-speed camera captures diffraction data in the form of a movie. The crystal structure is subsequently determined by using established X-ray crystallographic software. MicroED is a technique still under development, and hands-on expertise in sample preparation, data acquisition and processing is not always readily accessible. This comprehensive guide on MicroED sample preparation addresses commonly used methods for various sample categories, including room temperature solid-state small molecules and soluble and membrane protein crystals. Beyond detailing the steps of sample preparation for new users, and because every crystal requires unique growth and sample-preparation conditions, this resource provides instructions and optimization strategies for MicroED sample preparation. The protocol is suitable for users with expertise in biochemistry, crystallography, general cryo-EM and crystallography data processing. MicroED experiments, from sample vitrification to final structure, can take anywhere from one workday to multiple weeks, especially when cryogenic focused ion beam milling is involved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) has advanced structural methods across a range of sample types, from small molecules to proteins. This cryogenic electron microscopy (cryo-EM) technique involves the continuous rotation of small 3D crystals in the electron beam, while a high-speed camera captures diffraction data in the form of a movie. The crystal structure is subsequently determined by using established X-ray crystallographic software. MicroED is a technique still under development, and hands-on expertise in sample preparation, data acquisition and processing is not always readily accessible. This comprehensive guide on MicroED sample preparation addresses commonly used methods for various sample categories, including room temperature solid-state small molecules and soluble and membrane protein crystals. Beyond detailing the steps of sample preparation for new users, and because every crystal requires unique growth and sample-preparation conditions, this resource provides instructions and optimization strategies for MicroED sample preparation. The protocol is suitable for users with expertise in biochemistry, crystallography, general cryo-EM and crystallography data processing. MicroED experiments, from sample vitrification to final structure, can take anywhere from one workday to multiple weeks, especially when cryogenic focused ion beam milling is involved.
203.
Ruma, Yasmeen N.; Bu, Guanhong; Hattne, Johan; Gonen, Tamir
MicroED structure of the C11 cysteine protease clostripain
In: Journal of Structural Biology: X, vol. 10, pp. 100107, 2024, ISSN: 2590-1524.
@article{Ruma2024b,
title = {MicroED structure of the C11 cysteine protease clostripain},
author = {Yasmeen N. Ruma and Guanhong Bu and Johan Hattne and Tamir Gonen},
doi = {10.1016/j.yjsbx.2024.100107},
issn = {2590-1524},
year = {2024},
date = {2024-12-00},
urldate = {2024-12-00},
journal = {Journal of Structural Biology: X},
volume = {10},
pages = {100107},
publisher = {Elsevier BV},
abstract = {Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.
202.
Liu, Xiaoyu; Xia, Xian; Martynowycz, Michael W.; Gonen, Tamir; Zhou, Z. Hong
In: Nat Commun, vol. 15, no. 1, 2024, ISSN: 2041-1723.
@article{Liu2024,
title = {Molecular sociology of virus-induced cellular condensates supporting reovirus assembly and replication},
author = {Xiaoyu Liu and Xian Xia and Michael W. Martynowycz and Tamir Gonen and Z. Hong Zhou},
doi = {10.1038/s41467-024-54968-7},
issn = {2041-1723},
year = {2024},
date = {2024-12-00},
urldate = {2024-12-00},
journal = {Nat Commun},
volume = {15},
number = {1},
publisher = {Springer Science and Business Media LLC},
abstract = {Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) of focused ion beam (FIB) milling-produced lamellae of mammalian reovirus (MRV)-infected cells, we visualized the molecular organization and interplay (i.e., “molecular sociology”) of host and virus in 3D at two time points post-infection, enabling a detailed description of these condensates and a mechanistic understanding of MRV replication within them. Expanding over time, the condensate fashions host ribosomes at its periphery, and host microtubules, lipid membranes, and viral molecules in its interior, forming a 3D architecture that supports the dynamic processes of viral genome replication and capsid assembly. A total of six MRV assembly intermediates are identified inside the condensate: star core, empty and genome-containing cores, empty and full virions, and outer shell particle. Except for star core, these intermediates are visualized at atomic resolution by cryogenic electron microscopy (cryoEM) of cellular extracts. The temporal sequence and spatial rearrangement among these viral intermediates choreograph the viral life cycle within the condensates. Together, the molecular sociology of MRV-induced cellular condensate highlights the functional advantage of transient enrichment of molecules at the right location and time for viral replication.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) of focused ion beam (FIB) milling-produced lamellae of mammalian reovirus (MRV)-infected cells, we visualized the molecular organization and interplay (i.e., “molecular sociology”) of host and virus in 3D at two time points post-infection, enabling a detailed description of these condensates and a mechanistic understanding of MRV replication within them. Expanding over time, the condensate fashions host ribosomes at its periphery, and host microtubules, lipid membranes, and viral molecules in its interior, forming a 3D architecture that supports the dynamic processes of viral genome replication and capsid assembly. A total of six MRV assembly intermediates are identified inside the condensate: star core, empty and genome-containing cores, empty and full virions, and outer shell particle. Except for star core, these intermediates are visualized at atomic resolution by cryogenic electron microscopy (cryoEM) of cellular extracts. The temporal sequence and spatial rearrangement among these viral intermediates choreograph the viral life cycle within the condensates. Together, the molecular sociology of MRV-induced cellular condensate highlights the functional advantage of transient enrichment of molecules at the right location and time for viral replication.
201.
Lin, Jieye; Unge, Johan; Gonen, Tamir
bioRxiv, 2024.
@unpublished{Lin2024d,
title = {MicroED Structures of Fluticasone Furoate and Fluticasone Propionate Provide New Insights to Their Function},
author = {Jieye Lin and Johan Unge and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2024.09.18.613782},
doi = {10.1101/2024.09.18.613782},
year = {2024},
date = {2024-09-19},
urldate = {2024-09-19},
publisher = {Cold Spring Harbor Laboratory},
abstract = {The detailed understanding of fluticasone, a widely prescribed medicine for allergic rhinitis, asthma, and chronic obstructive pulmonary disease (COPD), has not been complete due to challenges in structural elucidation. The three-dimensional (3D) structure of fluticasone furoate1remained undetermined for decades, while the existing structures of fluticasone propionate 2 required refinement against improved data. In this study, we applied microcrystal electron diffraction (MicroED) to determine the 3D structures of 1 and 2in their drug formulation state. Density functional theory (DFT) calculations were utilized to model solvent effects to determine the preferred geometries in solution. A comparative analysis of structures of 1 and 2 across three states (drug formulation state, in solution, and biologically active state) revealed major conformational changes during the entire transition. Potential energy plots were calculated for the most dynamic bonds, uncovering their rotational barriers. This study underscores the combined use of MicroED and DFT calculations to provide a comprehensive understanding of conformational and energy changes during drug functioning in humans. The quantitative comparison highlights the subtle structural differences that can lead to significant functional changes in pharmaceutical properties.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
The detailed understanding of fluticasone, a widely prescribed medicine for allergic rhinitis, asthma, and chronic obstructive pulmonary disease (COPD), has not been complete due to challenges in structural elucidation. The three-dimensional (3D) structure of fluticasone furoate1remained undetermined for decades, while the existing structures of fluticasone propionate 2 required refinement against improved data. In this study, we applied microcrystal electron diffraction (MicroED) to determine the 3D structures of 1 and 2in their drug formulation state. Density functional theory (DFT) calculations were utilized to model solvent effects to determine the preferred geometries in solution. A comparative analysis of structures of 1 and 2 across three states (drug formulation state, in solution, and biologically active state) revealed major conformational changes during the entire transition. Potential energy plots were calculated for the most dynamic bonds, uncovering their rotational barriers. This study underscores the combined use of MicroED and DFT calculations to provide a comprehensive understanding of conformational and energy changes during drug functioning in humans. The quantitative comparison highlights the subtle structural differences that can lead to significant functional changes in pharmaceutical properties.
200.
Lin, Jieye; Bu, Guanhong; Unge, Johan; Gonen, Tamir
An Updated Structure of Oxybutynin Hydrochloride
In: Advanced Science, 2024, ISSN: 2198-3844.
@article{Lin2024c,
title = {An Updated Structure of Oxybutynin Hydrochloride},
author = {Jieye Lin and Guanhong Bu and Johan Unge and Tamir Gonen},
doi = {10.1002/advs.202406494},
issn = {2198-3844},
year = {2024},
date = {2024-09-03},
urldate = {2024-09-03},
journal = {Advanced Science},
publisher = {Wiley},
abstract = {Oxybutynin (Ditropan), a widely distributed muscarinic antagonist for treating the overactive bladder, has been awaiting a definitive crystal structure for ≈50 years due to the sample and technique limitations. Past reports used powder X‐ray diffraction (PXRD) to shed light on the possible packing of the molecule however their model showed some inconsistencies when compared with the 2D chemical structure. These are largely attributed to X‐ray‐induced photoreduction. Here microcrystal electron diffraction (MicroED) is used to successfully unveil the experimental 3D structure of oxybutynin hydrochloride showing marked improvement over the reported PXRD structure. Using the improved model, molecular docking is applied to investigate the binding mechanism between M3 muscarinic receptor (M3R) and (R)‐oxybutynin, revealing essential contacts/residues and conformational changes within the protein pocket. A possible universal conformation is proposed for M3R antagonists, which is valuable for future drug development and optimization. This study underscores the immense potential of MicroED as a complementary technique for elucidating unknown pharmaceutical structures, as well as for protein‐drug interactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Oxybutynin (Ditropan), a widely distributed muscarinic antagonist for treating the overactive bladder, has been awaiting a definitive crystal structure for ≈50 years due to the sample and technique limitations. Past reports used powder X‐ray diffraction (PXRD) to shed light on the possible packing of the molecule however their model showed some inconsistencies when compared with the 2D chemical structure. These are largely attributed to X‐ray‐induced photoreduction. Here microcrystal electron diffraction (MicroED) is used to successfully unveil the experimental 3D structure of oxybutynin hydrochloride showing marked improvement over the reported PXRD structure. Using the improved model, molecular docking is applied to investigate the binding mechanism between M3 muscarinic receptor (M3R) and (R)‐oxybutynin, revealing essential contacts/residues and conformational changes within the protein pocket. A possible universal conformation is proposed for M3R antagonists, which is valuable for future drug development and optimization. This study underscores the immense potential of MicroED as a complementary technique for elucidating unknown pharmaceutical structures, as well as for protein‐drug interactions.
199.
Clabbers, Max T. B.; Hattne, Johan; Martynowycz, Michael W.; Gonen, Tamir
Energy filtering enables macromolecular MicroED data at sub-atomic resolution
bioRxiv, 2024.
@unpublished{Clabbers2024,
title = {Energy filtering enables macromolecular MicroED data at sub-atomic resolution},
author = {Max T.B. Clabbers and Johan Hattne and Michael W. Martynowycz and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2024.08.29.610380},
doi = {10.1101/2024.08.29.610380},
year = {2024},
date = {2024-08-29},
urldate = {2024-08-29},
publisher = {Cold Spring Harbor Laboratory},
abstract = {High resolution information is important for accurate structure modelling. However, this level of detail is typically difficult to attain in macromolecular crystallography because the diffracted intensities rapidly fade with increasing resolution. The problem cannot be circumvented by increasing the fluence as this leads to detrimental radiation damage. Previously, we demonstrated that high quality MicroED data can be obtained at low flux conditions using electron counting with direct electron detectors. The improved sensitivity and accuracy of these detectors essentially eliminate the read-out noise, such that the measurement of faint high-resolution reflections is limited by other sources of noise. Inelastic scattering is a major contributor of such noise, increasing background counts and broadening diffraction spots. Here, we demonstrate that a substantial improvement in signal-to-noise ratio can be achieved using an energy filter to largely remove the inelastically scattered electrons. This strategy resulted in sub-atomic resolution MicroED data from proteinase K crystals, enabling accurate structure modelling and the visualization of detailed features. Interestingly, filtering out the noise revealed diffuse scattering phenomena that can hold additional structural information. Our findings suggest that combining energy filtering and electron counting can provide more accurate measurements at higher resolution, providing better insights into protein function and facilitating more precise model refinement.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
High resolution information is important for accurate structure modelling. However, this level of detail is typically difficult to attain in macromolecular crystallography because the diffracted intensities rapidly fade with increasing resolution. The problem cannot be circumvented by increasing the fluence as this leads to detrimental radiation damage. Previously, we demonstrated that high quality MicroED data can be obtained at low flux conditions using electron counting with direct electron detectors. The improved sensitivity and accuracy of these detectors essentially eliminate the read-out noise, such that the measurement of faint high-resolution reflections is limited by other sources of noise. Inelastic scattering is a major contributor of such noise, increasing background counts and broadening diffraction spots. Here, we demonstrate that a substantial improvement in signal-to-noise ratio can be achieved using an energy filter to largely remove the inelastically scattered electrons. This strategy resulted in sub-atomic resolution MicroED data from proteinase K crystals, enabling accurate structure modelling and the visualization of detailed features. Interestingly, filtering out the noise revealed diffuse scattering phenomena that can hold additional structural information. Our findings suggest that combining energy filtering and electron counting can provide more accurate measurements at higher resolution, providing better insights into protein function and facilitating more precise model refinement.
198.
Lin, Jieye; Bu, Guanhong; Unge, Johan; Gonen, Tamir
Uncovering the Elusive Structures and Mechanisms of Prevalent Antidepressants
In: Advanced Therapeutics, 2024, ISSN: 2366-3987.
@article{Lin2024e,
title = {Uncovering the Elusive Structures and Mechanisms of Prevalent Antidepressants},
author = {Jieye Lin and Guanhong Bu and Johan Unge and Tamir Gonen},
doi = {10.1002/adtp.202400117},
issn = {2366-3987},
year = {2024},
date = {2024-07-31},
urldate = {2024-07-31},
journal = {Advanced Therapeutics},
publisher = {Wiley},
abstract = {Most treatments to alleviate major depression work by either inhibiting human monoamine transporters, vital for the reuptake of monoamine neurotransmitters, or by inhibiting monoamine oxidases, which are vital for their degradation. The analysis of the experimental 3D structures of those antidepressants in their drug formulation state is key to precision drug design and development. In this study, microcrystal electron diffraction (MicroED) is applied to reveal the atomic 3D structures for the first time of five of the most prevalent antidepressants (reboxetine, pipofezine, ansofaxine, phenelzine, and bifemelane) directly from the commercially available powder of the active ingredients. Their modes of binding are investigated by molecular docking, revealing the essential contacts and conformational changes into the biologically active state. This study underscores the combined use of MicroED and molecular docking to uncover elusive drug structures and mechanisms to aid in further drug development pipelines.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Most treatments to alleviate major depression work by either inhibiting human monoamine transporters, vital for the reuptake of monoamine neurotransmitters, or by inhibiting monoamine oxidases, which are vital for their degradation. The analysis of the experimental 3D structures of those antidepressants in their drug formulation state is key to precision drug design and development. In this study, microcrystal electron diffraction (MicroED) is applied to reveal the atomic 3D structures for the first time of five of the most prevalent antidepressants (reboxetine, pipofezine, ansofaxine, phenelzine, and bifemelane) directly from the commercially available powder of the active ingredients. Their modes of binding are investigated by molecular docking, revealing the essential contacts and conformational changes into the biologically active state. This study underscores the combined use of MicroED and molecular docking to uncover elusive drug structures and mechanisms to aid in further drug development pipelines.
197.
Lin, Jieye; Bu, Guanhong; Unge, Johan; Gonen, Tamir
An Updated Structure of Oxybutynin Hydrochloride
bioRxiv, 2024.
@unpublished{Lin2024b,
title = {An Updated Structure of Oxybutynin Hydrochloride},
author = {Jieye Lin and Guanhong Bu and Johan Unge and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2024.06.05.597682},
doi = {10.1101/2024.06.05.597682},
year = {2024},
date = {2024-06-06},
urldate = {2024-06-06},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Oxybutynin (Ditropan), a widely distributed muscarinic antagonist for treating the overactive bladder, has been awaiting a definitive crystal structure for nearly 50 years due to the sample and technique limitations. Past reports used powder X-ray diffraction (PCRD) to shed light on the possible packing of the molecule however a 3D structure remained elusive. Here we used Microcrystal Electron Diffraction (MicroED) to successfully unveil the 3D structure of oxybutynin hydrochloride. We identify several inconsistencies between the reported PXRD analyses and the experimental structure. Using the improved model, molecular docking was applied to investigate the binding mechanism between M3muscarinic receptor (M3R) and (R)-oxybutynin, revealing essential contacts/residues and conformational changes within the protein pocket. A possible universal conformation was proposed for M3R antagonists, which is valuable for future drug development and optimization. This study underscores the immense potential of MicroED as a complementary technique for elucidating the unknown pharmaceutical crystal structures, as well as for the protein-drug interactions.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Oxybutynin (Ditropan), a widely distributed muscarinic antagonist for treating the overactive bladder, has been awaiting a definitive crystal structure for nearly 50 years due to the sample and technique limitations. Past reports used powder X-ray diffraction (PCRD) to shed light on the possible packing of the molecule however a 3D structure remained elusive. Here we used Microcrystal Electron Diffraction (MicroED) to successfully unveil the 3D structure of oxybutynin hydrochloride. We identify several inconsistencies between the reported PXRD analyses and the experimental structure. Using the improved model, molecular docking was applied to investigate the binding mechanism between M3muscarinic receptor (M3R) and (R)-oxybutynin, revealing essential contacts/residues and conformational changes within the protein pocket. A possible universal conformation was proposed for M3R antagonists, which is valuable for future drug development and optimization. This study underscores the immense potential of MicroED as a complementary technique for elucidating the unknown pharmaceutical crystal structures, as well as for the protein-drug interactions.
196.
Gillman, Cody; Bu, Guanhong; Danelius, Emma; Hattne, Johan; Nannenga, Brent L.; Gonen, Tamir
Eliminating the missing cone challenge through innovative approaches
In: Journal of Structural Biology: X, vol. 9, pp. 100102, 2024, ISSN: 2590-1524.
@article{Gillman2024b,
title = {Eliminating the missing cone challenge through innovative approaches},
author = {Cody Gillman and Guanhong Bu and Emma Danelius and Johan Hattne and Brent L. Nannenga and Tamir Gonen},
doi = {10.1016/j.yjsbx.2024.100102},
issn = {2590-1524},
year = {2024},
date = {2024-06-00},
urldate = {2024-06-00},
journal = {Journal of Structural Biology: X},
volume = {9},
pages = {100102},
publisher = {Elsevier BV},
abstract = {Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED.
195.
Nicolas, William J.; Shiriaeva, Anna; Martynowycz, Michael W.; Grey, Angus C; Ruma, Yasmeen; Donaldson, Paul J; Gonen, Tamir
Structure of the lens MP20 mediated adhesive junction
bioRxiv, 2024.
@unpublished{Nicolas2024,
title = {Structure of the lens MP20 mediated adhesive junction},
author = {William J. Nicolas and Anna Shiriaeva and Michael W. Martynowycz and Angus C Grey and Yasmeen Ruma and Paul J Donaldson and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2024.05.13.594022},
doi = {10.1101/2024.05.13.594022},
year = {2024},
date = {2024-05-14},
urldate = {2024-05-14},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C-termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C-termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency.
194.
Unge, Johan; Lin, Jieye; Weaver, Sara J; Her, Ampon Sae; Gonen, Tamir
Compositional Analysis of Complex Mixtures using Automatic MicroED Data Collection
In: Advanced Science, 2024, ISSN: 2198-3844.
@article{Unge2024,
title = {Compositional Analysis of Complex Mixtures using Automatic MicroED Data Collection},
author = {Johan Unge and Jieye Lin and Sara J Weaver and Ampon Sae Her and Tamir Gonen},
doi = {10.1002/advs.202400081},
issn = {2198-3844},
year = {2024},
date = {2024-04-22},
urldate = {2024-04-22},
journal = {Advanced Science},
publisher = {Wiley},
abstract = {Quantitative analysis of complex mixtures, including compounds having similar chemical properties, is demonstrated using an automatic and high throughput approach to microcrystal electron diffraction (MicroED). Compositional analysis of organic and inorganic compounds can be accurately executed without the need of diffraction standards. Additionally, with sufficient statistics, small amounts of compounds in mixtures can be reliably detected. These compounds can be distinguished by their crystal structure properties prior to structure solution. In addition, if the crystals are of good quality, the crystal structures can be generated on the fly, providing a complete analysis of the sample. 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 automatic and high throughput approach to MicroED, and with the use of SerialEM for data collection, data from thousands of crystals allow sufficient statistics to detect even small amounts of compounds reliably.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Quantitative analysis of complex mixtures, including compounds having similar chemical properties, is demonstrated using an automatic and high throughput approach to microcrystal electron diffraction (MicroED). Compositional analysis of organic and inorganic compounds can be accurately executed without the need of diffraction standards. Additionally, with sufficient statistics, small amounts of compounds in mixtures can be reliably detected. These compounds can be distinguished by their crystal structure properties prior to structure solution. In addition, if the crystals are of good quality, the crystal structures can be generated on the fly, providing a complete analysis of the sample. 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 automatic and high throughput approach to MicroED, and with the use of SerialEM for data collection, data from thousands of crystals allow sufficient statistics to detect even small amounts of compounds reliably.
193.
Xia, Xian; Sung, Po-Yu; Martynowycz, Michael W.; Gonen, Tamir; Roy, Polly; Zhou, Z. Hong
RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells
In: Cell, 2024, ISSN: 0092-8674.
@article{Xia2024,
title = {RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells},
author = {Xian Xia and Po-Yu Sung and Michael W. Martynowycz and Tamir Gonen and Polly Roy and Z. Hong Zhou},
doi = {10.1016/j.cell.2024.03.007},
issn = {0092-8674},
year = {2024},
date = {2024-04-00},
urldate = {2024-04-00},
journal = {Cell},
publisher = {Elsevier BV},
abstract = {Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed “duality” characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed “duality” characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.
192.
Bu, Guanhong; Danelius, Emma; Wieske, Lianne H. E.; Gonen, Tamir
Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED
In: Advanced Biology, vol. 8, iss. 5, pp. 2300570, 2024, ISSN: 2701-0198.
@article{Bu2024,
title = {Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED},
author = {Guanhong Bu and Emma Danelius and Lianne H.E. Wieske and Tamir Gonen},
doi = {10.1002/adbi.202300570},
issn = {2701-0198},
year = {2024},
date = {2024-02-21},
urldate = {2024-02-21},
journal = {Advanced Biology},
volume = {8},
issue = {5},
pages = {2300570},
publisher = {Wiley},
abstract = {Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus (HCV) infection. Its structures have been elusive to the public until recently when one of the crystal forms is solved by microcrystal electron diffraction (MicroED). In this work, the MicroED structures of two distinct polymorphic crystal forms of paritaprevir are reported from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropyl sulfonamide and methyl pyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the HCV non‐structural 3/4A (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 acyl sulfonamide inhibitors to the HCV NS3/4A serine protease. In addition, this also demonstrates the opportunity to derive different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus (HCV) infection. Its structures have been elusive to the public until recently when one of the crystal forms is solved by microcrystal electron diffraction (MicroED). In this work, the MicroED structures of two distinct polymorphic crystal forms of paritaprevir are reported from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropyl sulfonamide and methyl pyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the HCV non‐structural 3/4A (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 acyl sulfonamide inhibitors to the HCV NS3/4A serine protease. In addition, this also demonstrates the opportunity to derive different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.
191.
Gillman, Cody; Bu, Guanhong; Danelius, Emma; Hattne, Johan; Nannenga, Brent; Gonen, Tamir
Eliminating the Missing Cone Challenge through Innovative Approaches
bioRxiv, 2024.
@unpublished{Gillman2024,
title = {Eliminating the Missing Cone Challenge through Innovative Approaches},
author = {Cody Gillman and Guanhong Bu and Emma Danelius and Johan Hattne and Brent Nannenga and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2024.01.11.575283},
doi = {10.1101/2024.01.11.575283},
year = {2024},
date = {2024-01-14},
urldate = {2024-01-14},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If, as is typically the case, the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the grid cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that this method could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop crystallization approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the COVID-19 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting preferred orientations, unlocking new possibilities for structure determination by MicroED.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If, as is typically the case, the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the grid cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that this method could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop crystallization approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the COVID-19 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting preferred orientations, unlocking new possibilities for structure determination by MicroED.
190.
Ruma, Yasmeen N; Bu, Guanhong; Gonen, Tamir
MicroED structure of the C11 cysteine protease Clostripain
bioRxiv, 2024.
@unpublished{Ruma2024,
title = {MicroED structure of the C11 cysteine protease Clostripain},
author = {Yasmeen N Ruma and Guanhong Bu and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2024.01.04.574240},
doi = {10.1101/2024.01.04.574240},
year = {2024},
date = {2024-01-05},
urldate = {2024-01-05},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum Clostripain determined at 3.6 Angstrom resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of Clostripain shows a typical Clan CD α/β/α; sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 to 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as Clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum Clostripain determined at 3.6 Angstrom resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of Clostripain shows a typical Clan CD α/β/α; sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 to 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as Clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.
189.
Lin, Jieye; Bu, Guanhong; Unge, Johan; Gonen, Tamir
Uncovering the Elusive Structures and Mechanisms of Prevalent Antidepressants
bioRxiv, 2024.
@unpublished{Lin2024,
title = {Uncovering the Elusive Structures and Mechanisms of Prevalent Antidepressants},
author = {Jieye Lin and Guanhong Bu and Johan Unge and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/2024.01.04.574265},
doi = {10.1101/2024.01.04.574265},
year = {2024},
date = {2024-01-05},
urldate = {2024-01-05},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Most treatments to alleviate major depression work by either inhibiting human monoamine transporters, vital for the reuptake of monoamine neurotransmitters, or by inhibiting monoamine oxidases, which are vital for their degradation. The analysis of the experimental 3D structures of those antidepressants in their drug formulation state is key to precision drug design and development. In this study, we apply microcrystal electron diffraction (MicroED) to reveal the atomic 3D structures for the first time of five of the most prevalent antidepressants (reboxetine, pipofezine, ansofaxine, phenelzine, bifemelane) directly from the commercially available powder of the active ingredients. Their modes of binding are investigated by molecular docking, revealing the essential contacts and conformational changes into the biologically active state. This study underscores the combined use of MicroED and molecular docking to uncover elusive drug structures and mechanisms to aid in further drug development pipelines.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Most treatments to alleviate major depression work by either inhibiting human monoamine transporters, vital for the reuptake of monoamine neurotransmitters, or by inhibiting monoamine oxidases, which are vital for their degradation. The analysis of the experimental 3D structures of those antidepressants in their drug formulation state is key to precision drug design and development. In this study, we apply microcrystal electron diffraction (MicroED) to reveal the atomic 3D structures for the first time of five of the most prevalent antidepressants (reboxetine, pipofezine, ansofaxine, phenelzine, bifemelane) directly from the commercially available powder of the active ingredients. Their modes of binding are investigated by molecular docking, revealing the essential contacts and conformational changes into the biologically active state. This study underscores the combined use of MicroED and molecular docking to uncover elusive drug structures and mechanisms to aid in further drug development pipelines.
2023
188.
Lin, Jieye; Unge, Johan; Gonen, Tamir
Unraveling the Structure of Meclizine Dihydrochloride with MicroED
In: Adv Sci (Weinh), pp. e2306435, 2023, ISSN: 2198-3844.
@article{pmid38044280,
title = {Unraveling the Structure of Meclizine Dihydrochloride with MicroED},
author = {Jieye Lin and Johan Unge and Tamir Gonen},
doi = {10.1002/advs.202306435},
issn = {2198-3844},
year = {2023},
date = {2023-12-01},
journal = {Adv Sci (Weinh)},
pages = {e2306435},
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. Single-crystal X-ray diffraction (SC-XRD) is previously unsuccessful for meclizine. Today, microcrystal electron diffraction (MicroED) enables the analysis of nano- or micro-sized crystals that are merely a billionth the size needed for SC-XRD directly from seemingly amorphous powder. In this study, MicroED to determine the 3D crystal structure of meclizine dihydrochloride is used. Two racemic enantiomers (R/S) are found in the unit cell, which is packed as repetitive double layers in the crystal lattice. The packing is made of multiple strong N-H-Cl hydrogen bonding interactions and weak interactions like C-H-Cl and pi-stacking. Molecular docking reveals 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) shows the conserved binding sites. This research illustrates the combined use of MicroED and molecular docking in unraveling elusive drug structures and protein-drug interactions for precision drug design and optimization.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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. Single-crystal X-ray diffraction (SC-XRD) is previously unsuccessful for meclizine. Today, microcrystal electron diffraction (MicroED) enables the analysis of nano- or micro-sized crystals that are merely a billionth the size needed for SC-XRD directly from seemingly amorphous powder. In this study, MicroED to determine the 3D crystal structure of meclizine dihydrochloride is used. Two racemic enantiomers (R/S) are found in the unit cell, which is packed as repetitive double layers in the crystal lattice. The packing is made of multiple strong N-H-Cl hydrogen bonding interactions and weak interactions like C-H-Cl and pi-stacking. Molecular docking reveals 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) shows the conserved binding sites. This research illustrates the combined use of MicroED and molecular docking in unraveling elusive drug structures and protein-drug interactions for precision drug design and optimization.
187.
Walsh, Martin A; Nannenga, Brent L; Gonen, Tamir; Sexton, Patrick M; Wootten, Denise; Chiu, Wah; Sun, Fei; Carragher, Bridget; Potter, Clinton S; Agard, David; Burley, Stephen K
The next decade in structural biology
In: Structure, vol. 31, no. 11, pp. 1284–1288, 2023, ISSN: 1878-4186.
@article{pmid37922863,
title = {The next decade in structural biology},
author = {Martin A Walsh and Brent L Nannenga and Tamir Gonen and Patrick M Sexton and Denise Wootten and Wah Chiu and Fei Sun and Bridget Carragher and Clinton S Potter and David Agard and Stephen K Burley},
doi = {10.1016/j.str.2023.10.002},
issn = {1878-4186},
year = {2023},
date = {2023-11-01},
journal = {Structure},
volume = {31},
number = {11},
pages = {1284--1288},
abstract = {As we celebrate the 30 anniversary of Structure, we have asked structural biologists about their expectations on how their respective fields are likely to develop in the next ten years in this collection of Voices.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
As we celebrate the 30 anniversary of Structure, we have asked structural biologists about their expectations on how their respective fields are likely to develop in the next ten years in this collection of Voices.
186.
Danelius, Emma; Bu, Guanhong; Wieske, Lianne H E; Gonen, Tamir
In: ACS Chem Biol, 2023, ISSN: 1554-8937.
@article{pmid37944119,
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 H E Wieske and Tamir Gonen},
doi = {10.1021/acschembio.3c00611},
issn = {1554-8937},
year = {2023},
date = {2023-11-01},
journal = {ACS Chem Biol},
abstract = {Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as to 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 that 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 an impact on contemporary drug discovery as well as natural product exploration.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as to 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 that 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 an impact on contemporary drug discovery as well as natural product exploration.
185.
Hattne, Johan; Clabbers, Max T. B.; Martynowycz, Michael W.; Gonen, Tamir
Electron counting with direct electron detectors in MicroED
In: Structure, 2023, ISSN: 0969-2126.
@article{Hattne2023b,
title = {Electron counting with direct electron detectors in MicroED},
author = {Johan Hattne and Max T.B. Clabbers and Michael W. Martynowycz and Tamir Gonen},
doi = {10.1016/j.str.2023.10.011},
issn = {0969-2126},
year = {2023},
date = {2023-11-00},
urldate = {2023-11-00},
journal = {Structure},
publisher = {Elsevier BV},
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 number of electrons used for data collection. 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 fluence also alleviates concerns about radiation damage which limits the information that can be recovered from a diffraction measurement. The major concern with electron-counting direct detectors lies at the low end of the resolution spectrum: their limited linear range makes strong low-resolution reflections susceptible to coincidence loss and careful data collection is required to avoid compromising data quality. Nevertheless, these cameras 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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 number of electrons used for data collection. 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 fluence also alleviates concerns about radiation damage which limits the information that can be recovered from a diffraction measurement. The major concern with electron-counting direct detectors lies at the low end of the resolution spectrum: their limited linear range makes strong low-resolution reflections susceptible to coincidence loss and careful data collection is required to avoid compromising data quality. Nevertheless, these cameras 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.
184.
Lin, Jieye; Unge, Johan; Gonen, Tamir
Distinct Conformations of Mirabegron Determined by MicroED
In: Adv Sci (Weinh), pp. e2304476, 2023, ISSN: 2198-3844.
@article{pmid37847906,
title = {Distinct Conformations of Mirabegron Determined by MicroED},
author = {Jieye Lin and Johan Unge and Tamir Gonen},
doi = {10.1002/advs.202304476},
issn = {2198-3844},
year = {2023},
date = {2023-10-01},
urldate = {2023-10-01},
journal = {Adv Sci (Weinh)},
pages = {e2304476},
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, the authors employed microcrystal electron diffraction (MicroED) to reveal its elusive three-dimensional (3D) structure. They 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 are 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, 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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, the authors employed microcrystal electron diffraction (MicroED) to reveal its elusive three-dimensional (3D) structure. They 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 are 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, 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.
183.
Bai, Xiao-Chen; Gonen, Tamir; Gronenborn, Angela M; Perrakis, Anastassis; Thorn, Andrea; Yang, Jianyi
Challenges and opportunities in macromolecular structure determination
In: Nat Rev Mol Cell Biol, 2023, ISSN: 1471-0080.
@article{pmid37848590,
title = {Challenges and opportunities in macromolecular structure determination},
author = {Xiao-Chen Bai and Tamir Gonen and Angela M Gronenborn and Anastassis Perrakis and Andrea Thorn and Jianyi Yang},
doi = {10.1038/s41580-023-00659-y},
issn = {1471-0080},
year = {2023},
date = {2023-10-01},
journal = {Nat Rev Mol Cell Biol},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
182.
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}
}
181.
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}
}
180.
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},
url = {https://cryoem.ucla.edu/wp-content/uploads/2023_Nannenga-Gonen.pdf, Main text},
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.
179.
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.
178.
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},
urldate = {2023-08-00},
journal = {Structure},
publisher = {Elsevier BV},
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)2 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)2 DNA, demonstrating the successful application of cryo-FIB milling and MicroED to the investigation of nucleic acid crystals.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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)2 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)2 DNA, demonstrating the successful application of cryo-FIB milling and MicroED to the investigation of nucleic acid crystals.
177.
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.
176.
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}
}
175.
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.
174.
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.
173.
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.
172.
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.
171.
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.
170.
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}
}
169.
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.
168.
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 = {<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>},
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>
167.
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}
}
166.
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.
165.
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
164.
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.
163.
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.
162.
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.
161.
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.
160.
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}
}
159.
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.
158.
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.
157.
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.
156.
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.
155.
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.