CV for Tamir Gonen, PhD, MRSNZ

@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}

}

@article{pmid35371502,

title = {MicroED: conception, practice and future opportunities},

author = {Max T B Clabbers and Anna Shiriaeva and Tamir Gonen},

doi = {10.1107/S2052252521013063},

issn = {2052-2525},

year  = {2022},

date = {2022-03-01},

urldate = {2022-03-01},

journal = {IUCrJ},

volume = {9},

number = {Pt 2},

pages = {169--179},

abstract = {This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today's application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35191473,

title = {Studying membrane proteins with MicroED},

author = {Marc J Gallenito and Tamir Gonen},

doi = {10.1042/BST20210911},

issn = {1470-8752},

year  = {2022},

date = {2022-02-28},

urldate = {2022-02-01},

journal = {Biochem Soc Trans},

volume = {50},

number = {1},

pages = {231--239},

abstract = {The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34873060,

title = {Benchmarking the ideal sample thickness in cryo-EM},

author = {Michael W Martynowycz and Max T B Clabbers and Johan Unge and Johan Hattne and Tamir Gonen},

doi = {10.1073/pnas.2108884118},

issn = {1091-6490},

year  = {2021},

date = {2021-12-01},

urldate = {2021-12-01},

journal = {Proc Natl Acad Sci U S A},

volume = {118},

number = {49},

abstract = {The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Clark2021,

title = {MicroED for the study of protein–ligand interactions and the potential for drug discovery},

author = {Lisa J. Clark and Guanhong Bu and Brent L. Nannenga and Tamir Gonen},

doi = {10.1038/s41570-021-00332-y},

year  = {2021},

date = {2021-10-27},

urldate = {2021-10-27},

journal = {Nat Rev Chem},

volume = {5},

pages = {853–858},

abstract = {Microcrystal electron diffraction (MicroED) is an electron cryo-microscopy (cryo-EM) technique used to determine molecular structures with crystals that are a millionth the size needed for traditional single-crystal X-ray crystallography. An exciting use of MicroED is in drug discovery and development, where it can be applied to the study of proteins and small molecule interactions, and for structure determination of natural products. The structures are then used for rational drug design and optimization. In this Perspective, we discuss the current applications of MicroED for structure determination of protein–ligand complexes and potential future applications in drug discovery.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34382014,

title = {Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED)},

author = {Michael W Martynowycz and Tamir Gonen},

doi = {10.1016/j.xpro.2021.100686},

issn = {2666-1667},

year  = {2021},

date = {2021-09-17},

journal = {STAR Protoc},

volume = {2},

number = {3},

pages = {100686},

abstract = {We present an in-depth protocol to reproducibly prepare crystalline lamellae from protein crystals for subsequent microcrystal electron diffraction (MicroED) experiments. This protocol covers typical soluble proteins and membrane proteins embedded in dense media. Following these steps will allow the user to prepare crystalline lamellae for protein structure determination by MicroED. For complete details on the use and execution of this protocol, please refer to Martynowycz et al. (2019a, 2020a).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{2020_Martynowycz,

title = {MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP},

author = {Martynowycz, Michael W. and Shiriaeva, Anna and Ge, Xuanrui and Hattne, Johan and Nannenga, Brent L. and Cherezov, Vadim and Gonen, Tamir},

doi = {10.1073/pnas.2106041118},

year  = {2021},

date = {2021-09-07},

urldate = {2021-09-07},

journal = {Proc Natl Acad Sci U S A},

volume = {118},

number = {36},

pages = {e2106041118},

organization = {bioRxiv},

abstract = {G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A2A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34153215,

title = {An Overview of Microcrystal Electron Diffraction (MicroED)},

author = {Xuelang Mu and Cody Gillman and Chi Nguyen and Tamir Gonen},

doi = {10.1146/annurev-biochem-081720-020121},

issn = {1545-4509},

year  = {2021},

date = {2021-06-20},

urldate = {2021-06-00},

journal = {Annu Rev Biochem},

volume = {90},

pages = {431--450},

abstract = {The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lei2020,

title = {A conformational change in the N Terminus of SLC38A9 signals mTORC1 activation},

author = {Hsiang-Ting Lei and Xuelang Mu and Johan Hattne and Tamir Gonen},

doi = {10.1016/j.str.2020.11.014},

year  = {2021},

date = {2021-05-06},

urldate = {2021-05-06},

journal = {Structure},

volume = {29},

number = {5},

pages = {426--432.e8},

abstract = {mTORC1 is a central hub that integrates environmental cues, such as cellular stresses and nutrient availability to modulate metabolism and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, emerged as a sensor for luminal arginine and as an activator of mTORC1. The amino acid-mediated activation of mTORC1 is regulated by the N-terminal domain of SLC38A9. Here, we determined the crystal structure of zebrafish SLC38A9 (drSLC38A9) and found the N-terminal fragment inserted deep within the transporter, bound in the substrate-binding pocket where normally arginine would bind. This represents a significant conformational change of the N-terminal domain (N-plug) when compared with our recent arginine-bound structure of drSLC38A9. We propose a ball-and-chain model for mTORC1 activation, where N-plug insertion and Rag GTPase binding with SLC38A9 is regulated by luminal arginine levels. This work provides important insights into nutrient sensing by SLC38A9 to activate the mTORC1 pathways in response to dietary amino acids.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33779618,

title = {Microcrystal Electron Diffraction of Small Molecules},

author = {M W Martynowycz and T Gonen},

doi = {10.3791/62313},

year  = {2021},

date = {2021-03-15},

journal = {J Vis Exp},

number = {169},

abstract = {A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{2021_Halaby,

title = {Microcrystal Electron Diffraction for Molecular Design of Functional Non-Fullerene Acceptor Structures},

author = {Steve Halaby and Michael W. Martynowycz and Ziyue Zhu and Sergei Tretiak and Andriy Zhugayevych and Tamir Gonen and and Martin Seifrid},

doi = {10.1021/acs.chemmater.0c04111},

year  = {2021},

date = {2021-01-25},

journal = {Chem. Mater.},

volume = {33},

number = {3},

pages = {966--977},

abstract = {Understanding the relationship between molecular structure and solid-state arrangement informs about the design of new organic semiconductor (OSC) materials with improved optoelectronic properties. However, determining their atomic structure remains challenging. Here, we report the lattice organization of two non-fullerene acceptors (NFAs) determined using microcrystal electron diffraction (MicroED) from crystals not tractable by X-ray crystallography. The MicroED structure of o-IDTBR was determined from a powder without crystallization, and a new polymorph of ITIC-Th is identified with the most distorted backbone of any NFA. Electronic structure calculations elucidate the relationships between molecular structures, lattice arrangements, and charge-transport properties for a number of NFA lattices. The high dimensionality of the connectivity of the 3D wire mesh topology is the best for robust charge transport within NFA crystals. However, some examples suffer from uneven electronic coupling. MicroED combined with advanced electronic structure modeling is a powerful new approach for structure determination, exploring polymorphism and guiding the design of new OSCs and NFAs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{2020a_Martynowycz,

title = {Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking},

author = {Michael W. Martynowycz and Tamir Gonen},

doi = {10.1016/j.str.2020.09.003},

year  = {2021},

date = {2021-01-07},

urldate = {2021-01-07},

journal = {Structure},

volume = {29},

number = {1},

pages = {88--95.e2},

organization = {bioRxiv},

abstract = {A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of approximately 200 nm using a focused ion beam, and MicroED data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Both the number of ligands bound and their occupancy is higher using on-grid soaking of microcrystals compared with much larger crystals treated similarly and investigated by X-ray crystallography. These results indicate that on-grid soaking ligands into microcrystals results in efficient uptake of ligands into protein microcrystals.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33293416,

title = {MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel},

author = {M W Martynowycz and F Khan and J Hattne and J Abramson and T Gonen},

doi = {10.1073/pnas.2020010117},

year  = {2020},

date = {2020-12-22},

journal = {Proc Natl Acad Sci U S A},

volume = {117},

number = {51},

pages = {32380--32385},

abstract = {A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{2020_Zhu,

title = {Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED},

author = {Lan Zhu and Guanhong Bu and Liang Jing and Dan Shi and Ming-Yue Lee and Tamir Gonen and Wei Liu and Brent L. Nannenga},

url = {https://cryoem.ucla.edu/wp-content/uploads/2020_Zhu.pdf, Main text},

doi = {https://doi.org/10.1016/j.str.2020.07.006},

year  = {2020},

date = {2020-10-06},

journal = {Structure},

volume = {28},

number = {10},

pages = {1149--1159.e4},

abstract = {The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A2A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Nannenga2020,

title = {Microcrystal electron diffraction methodology and applications},

author = {Brent L. Nannenga and Tamir Gonen},

doi = {10.1557/mrs.2019.287},

year  = {2020},

date = {2020-09-27},

journal = {MRS Bulletin},

volume = {44},

pages = {956--960},

abstract = {Microcrystal electron diffraction (MicroED) is a cryo-electron microscopy technique that utilizes three-dimensional nano- and microcrystals for high-resolution structure determination. These extremely small crystals are several orders of magnitude smaller than what is used in conventional x-ray diffraction experiments. MicroED is capable of providing high-quality data from samples that would otherwise be considered useless for diffraction measurements. Since its initial implementation, MicroED has been used in the fields of structural biology, chemistry, and materials science. In this article, we provide an overview of the MicroED methodology as well as examples of how MicroED in cryo-electron microscopy has been used for structure determination of a variety of samples.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32939523,

title = {MicroED in natural product and small molecule research},

author = {E Danelius and S Halaby and W A van der Donk and T Gonen},

doi = {10.1039/d0np00035c},

year  = {2020},

date = {2020-09-17},

urldate = {2020-09-20},

journal = {Nat Prod Rep},

volume = {38},

number = {3},

abstract = {The electron cryo-microscopy (cryo-EM) method Microcrystal Electron Diffraction (MicroED) allows the collection of high-resolution structural data from vanishingly small crystals that appear like amorphous powders or very fine needles. Since its debut in 2013, data collection and analysis schemes have been fine-tuned, and there are currently close to 100 structures determined by MicroED. Although originally developed to study proteins, MicroED is also very powerful for smaller systems, with some recent and very promising examples from the field of natural products. Herein, we review what has been achieved so far and provide examples of natural product structures, as well as demonstrate the expected future impact of MicroED to the field of natural product and small molecule research.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{2020_Richards,

title = {Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER},

author = {Logan S. Richards and Claudia Millán and Jennifer Miao and Michael W. Martynowycz and Michael R. Sawaya and Tamir Gonen and Rafael J. Borges and Isabel Usón and Jose A. Rodriguez},

url = {https://cryoem.ucla.edu/wp-content/uploads/2020_Richards.pdf, Main text},

doi = {10.1107/S2059798320008049},

year  = {2020},

date = {2020-07-27},

journal = {Acta Crystallogr D Biol Crystallogr.},

volume = {76},

number = {8},

pages = {703-712},

abstract = {Structure determination of novel biological macromolecules by X-ray crystallo­graphy can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{2020_Nguyen,

title = {Beyond protein structure determination with MicroED},

author = {Chi Nguyen and Tamir Gonen},

url = {https://cryoem.ucla.edu/wp-content/uploads/2020_NguyenGonen.pdf, Main text},

doi = {10.1016/j.sbi.2020.05.018},

year  = {2020},

date = {2020-06-28},

urldate = {2020-06-28},

journal = {Curr. Opin. Struct. Biol.},

volume = {64},

pages = {51–58},

abstract = {Microcrystal electron diffraction (MicroED) was first coined and developed in 2013 at the Janelia Research Campus as a new modality in electron cryomicroscopy (cryoEM). Since then, MicroED has not only made important contributions in pushing the resolution limits of cryoEM protein structure characterization but also of peptides, small-organic and inorganic molecules, and natural-products that have resisted structure determination by other methods. This review showcases important recent developments in MicroED, highlighting the importance of the technique in fields of studies beyond protein structure determination where MicroED is beginning to have paradigm shifting roles.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32023481,

title = {Experimental Phasing of MicroED Data Using Radiation Damage},

author = {M W Martynowycz and J Hattne and T Gonen},

doi = {10.1016/j.str.2020.01.008},

year  = {2020},

date = {2020-04-07},

urldate = {2020-04-07},

journal = {Structure},

volume = {28},

number = {4},

pages = {458--464},

abstract = {We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present an example of an experimentally phased structure using only MicroED data. The structure of a seven-residue peptide is solved starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous-rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a "low-dose dataset." The data from the second pass were similarly merged to form a "damaged dataset." Differences between these two datasets were used to identify a single heavy-atom site from a Patterson difference map, and initial phases were generated. Finally, the structure was completed by iterative cycles of modeling and refinement.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32148858,

title = {Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals},

author = {A M Wolff and I D Young and R G Sierra and A S Brewster and M W Martynowycz and E Nango and M Sugahara and T Nakane and K Ito and A Aquila and A Bhowmick and J T Biel and S Carbajo and A E Cohen and S Cortez and A Gonzalez and T Hino and D Im and J D Koralek and M Kubo and T S Lazarou and T Nomura and S Owada and A J Samelson and T Tanaka and R Tanaka and E M Thompson and H van den Bedem and R A Woldeyes and F Yumoto and W Zhao and K Tono and S Boutet and S Iwata and T Gonen and N K Sauter and J S Fraser and M C Thompson},

doi = {10.1107/S205225252000072X},

year  = {2020},

date = {2020-03-01},

journal = {IUCrJ},

volume = {7},

number = {Pt 2},

pages = {306--323},

abstract = {Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31747522,

title = {Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation},

author = {Markus Dick and Nicholas S Sarai and Michael W Martynowycz and Tamir Gonen and Frances H Arnold},

doi = {10.1021/jacs.9b09864},

year  = {2019},

date = {2019-11-01},

journal = {J. Am. Chem. Soc.},

abstract = {We previously engineered the tryptophan synthase β-subunit (TrpB), which catalyzes the condensation of L-serine and indole to L-tryptophan, to synthesize a range of noncanonical amino acids from L-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds by attacking N1 of the substrate. Protecting N1 encouraged evolution towards C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pfquat with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereo-center at the β-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the β-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pfquat offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31612856,

title = {Structure-based inhibitors of amyloid beta core suggest a common interface with tau},

author = {Sarah L Griner and Paul Seidler and Jeannette Bowler and Kevin A Murray and Tianxiao Peter Yang and Shruti Sahay and Michael R Sawaya and Duilio Cascio and Jose A Rodriguez and Stephan Philipp and Justyna Sosna and Charles G Glabe and Tamir Gonen and David S Eisenberg},

doi = {10.7554/eLife.46924.001},

year  = {2019},

date = {2019-10-15},

urldate = {2019-01-01},

journal = {Elife},

volume = {8},

abstract = {Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31422911,

title = {Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED},

author = {Michael W Martynowycz and Wei Zhao and Johan Hattne and Grant J Jensen and Tamir Gonen},

doi = {10.1016/j.str.2019.07.004},

year  = {2019},

date = {2019-10-01},

journal = {Structure},

volume = {27},

number = {10},

pages = {1594--1600},

abstract = {Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31576224,

title = {MicroED with the Falcon III direct electron detector},

author = {Johan Hattne and Michael W Martynowycz and Pawel A Penczek and Tamir Gonen},

doi = {10.1107/S2052252519010583},

year  = {2019},

date = {2019-09-01},

journal = {IUCrJ},

volume = {6},

number = {Pt 5},

pages = {921--926},

abstract = {Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31350392,

title = {Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface},

author = {Rebeccah A Warmack and David R Boyer and Chih-Te Zee and Logan S Richards and Michael R Sawaya and Duilio Cascio and Tamir Gonen and David S Eisenberg and Steven G Clarke},

doi = {10.1038/s41467-019-11183-z},

year  = {2019},

date = {2019-07-26},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {3357},

abstract = {Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31320540,

title = {Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products},

author = {Chi P Ting and Michael A Funk and Steve L Halaby and Zhengan Zhang and Tamir Gonen and Wilfred A van der Donk},

doi = {10.1126/science.aau6232},

year  = {2019},

date = {2019-07-19},

journal = {Science},

volume = {365},

number = {6450},

pages = {280--284},

abstract = {Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA-dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid-derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid-derived natural products.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30986656,

title = {MicroED data collection with SerialEM},

author = {Jason M de la Cruz and Michael W Martynowycz and Johan Hattne and Tamir Gonen},

doi = {10.1016/j.ultramic.2019.03.009},

year  = {2019},

date = {2019-06-01},

journal = {Ultramicroscopy},

volume = {201},

pages = {77--80},

abstract = {The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31040436,

title = {The cryo-EM method microcrystal electron diffraction (MicroED)},

author = {Brent L Nannenga and Tamir Gonen},

doi = {10.1038/s41592-019-0395-x},

year  = {2019},

date = {2019-04-29},

journal = {Nat. Methods},

volume = {16},

number = {5},

pages = {369--379},

abstract = {In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30661853,

title = {Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size},

author = {Michael W Martynowycz and Wei Zhao and Johan Hattne and Grant J Jensen and Tamir Gonen},

doi = {10.1016/j.str.2018.12.003},

year  = {2019},

date = {2019-03-05},

urldate = {2019-03-01},

journal = {Structure},

volume = {27},

number = {3},

pages = {545--548},

abstract = {Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30867917,

title = {Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ},

author = {Chih-Te Zee and Calina Glynn and Marcus Gallagher-Jones and Jennifer Miao and Carlos G Santiago and Duilio Cascio and Tamir Gonen and Michael R Sawaya and Jose A Rodriguez},

doi = {10.1107/S2052252518017621},

year  = {2019},

date = {2019-03-01},

journal = {IUCrJ},

volume = {6},

number = {Pt 2},

pages = {197--205},

abstract = {The ice-nucleation protein InaZ from Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. Ab initio structures of this repeat segment, resolved to 1.1 Å by microfocus X-ray crystallography and to 0.9 Å by the cryo-EM method MicroED, were determined from both racemic and homochiral crystals. The benefits of racemic protein crystals for structure determination by MicroED were evaluated and it was confirmed that the phase restriction introduced by crystal centrosymmetry increases the number of successful trials during the ab initio phasing of the electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel β-sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all-left-handed sheets mate with their all-right-handed counterparts. This determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with submicrometre-sized crystals.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30659158,

title = {Structural basis for substrate binding and specificity of a sodium-alanine symporter AgcS},

author = {Jinming Ma and Hsiang-Ting Lei and Francis E Reyes and Silvia Sanchez-Martinez and Maen F Sarhan and Johan Hattne and Tamir Gonen},

doi = {10.1073/pnas.1806206116},

year  = {2019},

date = {2019-01-18},

journal = {Proc. Natl. Acad. Sci. U.S.A.},

volume = {116},

number = {6},

pages = {2086--2090},

abstract = {The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30530702,

title = {MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat},

author = {Michael D Purdy and Dan Shi and Jakub Chrustowicz and Johan Hattne and Tamir Gonen and Mark Yeager},

doi = {10.1073/pnas.1806806115},

year  = {2018},

date = {2018-12-26},

journal = {Proc. Natl. Acad. Sci. U.S.A.},

volume = {115},

number = {52},

pages = {13258--13263},

abstract = {HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30555912,

title = {The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination},

author = {Christopher G Jones and Michael W Martynowycz and Johan Hattne and Tyler J Fulton and Brian M Stoltz and Jose A Rodriguez and Hosea M Nelson and Tamir Gonen},

doi = {10.1021/acscentsci.8b00760},

year  = {2018},

date = {2018-11-02},

journal = {ACS Cent Sci},

volume = {4},

number = {11},

pages = {1587--1592},

abstract = {In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule's structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10-15 g) resulting in atomic resolution (<1 Å) crystal structures in minutes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}