CV for Tamir Gonen, PhD, MRSNZ

@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}

}

@article{pmid29872228,

title = {Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state},

author = {Hsiang-Ting Lei and Jinming Ma and Silvia Sanchez Martinez and Tamir Gonen},

doi = {10.1038/s41594-018-0072-2},

year  = {2018},

date = {2018-06-05},

journal = {Nat. Struct. Mol. Biol.},

volume = {25},

number = {6},

pages = {522--527},

abstract = {Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30167468,

title = {MicroED structure of the NaK ion channel reveals a Na⁺ partition process into the selectivity filter},

author = {Shian Liu and Tamir Gonen},

doi = {10.1038/s42003-018-0040-8},

year  = {2018},

date = {2018-05-03},

journal = {Commun Biol},

volume = {1},

pages = {38},

abstract = {Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29706530,

title = {Analysis of Global and Site-Specific Radiation Damage in Cryo-EM},

author = {Johan Hattne and Dan Shi and Calina Glynn and Chih-Te Zee and Marcus Gallagher-Jones and Michael W Martynowycz and Jose A Rodriguez and Tamir Gonen},

doi = {10.1016/j.str.2018.03.021},

year  = {2018},

date = {2018-05-01},

journal = {Structure},

volume = {26},

number = {5},

pages = {759--766},

abstract = {Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29531287,

title = {Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2},

author = {Elizabeth L Guenther and Peng Ge and Hamilton Trinh and Michael R Sawaya and Duilio Cascio and David R Boyer and Tamir Gonen and Hong Z Zhou and David S Eisenberg},

doi = {10.1038/s41594-018-0045-5},

year  = {2018},

date = {2018-03-12},

journal = {Nat. Struct. Mol. Biol.},

volume = {25},

number = {4},

pages = {311--319},

abstract = {Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29507202,

title = {Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system},

author = {Yuxi Liu and Shane Gonen and Tamir Gonen and Todd O Yeates},

doi = {10.1073/pnas.1718825115},

year  = {2018},

date = {2018-03-05},

urldate = {2018-03-27},

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

volume = {115},

number = {13},

pages = {3362--3367},

abstract = {Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30166936,

title = {From electron crystallography of 2D crystals to MicroED of 3D crystals},

author = {Michael W Martynowycz and Tamir Gonen},

doi = {10.1016/j.cocis.2018.01.010},

year  = {2018},

date = {2018-03-01},

journal = {Curr Opin Colloid Interface Sci},

volume = {34},

pages = {9--16},

abstract = {Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29282295,

title = {Common fibrillar spines of amyloid-β and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors},

author = {Pascal Krotee and Sarah L Griner and Michael R Sawaya and Duilio Cascio and Jose A Rodriguez and Dan Shi and Stephan Philipp and Kevin Murray and Lorena Saelices and Ji Lee and Paul Seidler and Charles G Glabe and Lin Jiang and Tamir Gonen and David S Eisenberg},

doi = {10.1074/jbc.M117.806109},

year  = {2018},

date = {2018-02-23},

urldate = {2017-02-23},

journal = {J. Biol. Chem.},

volume = {293},

number = {8},

pages = {2888--2902},

abstract = {Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aβ and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aβ(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aβ and hIAPP.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29439243,

title = {Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks},

author = {Michael P Hughes and Michael R Sawaya and David R Boyer and Lukasz Goldschmidt and Jose A Rodriguez and Duilio Cascio and Lisa Chong and Tamir Gonen and David S Eisenberg},

doi = {10.1126/science.aan6398},

year  = {2018},

date = {2018-02-09},

journal = {Science},

volume = {359},

number = {6376},

pages = {698--701},

abstract = {Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30167465,

title = {MicroED: a versatile cryoEM method for structure determination},

author = {Brent L Nannenga and Tamir Gonen},

doi = {10.1042/ETLS20170082},

year  = {2018},

date = {2018-02-06},

journal = {Emerg Top Life Sci},

volume = {2},

number = {1},

pages = {1--8},

abstract = {Micro-electron diffraction, or MicroED, is a structure determination method that uses a cryo-transmission electron microscope to collect electron diffraction data from nanocrystals. This technique has been successfully used to determine the high-resolution structures of many targets from crystals orders of magnitude smaller than what is needed for X-ray diffraction experiments. In this review, we will describe the MicroED method and recent structures that have been determined. Additionally, applications of electron diffraction to the fields of small molecule crystallography and materials science will be discussed.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29335561,

title = {Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp},

author = {Marcus Gallagher-Jones and Calina Glynn and David R Boyer and Michael W Martynowycz and Evelyn Hernandez and Jennifer Miao and Chih-Te Zee and Irina V Novikova and Lukasz Goldschmidt and Heather T McFarlane and Gustavo F Helguera and James E Evans and Michael R Sawaya and Duilio Cascio and David S Eisenberg and Tamir Gonen and Jose A Rodriguez},

doi = {10.1038/s41594-017-0018-0},

year  = {2018},

date = {2018-01-15},

journal = {Nat. Struct. Mol. Biol.},

volume = {25},

number = {2},

pages = {131--134},

abstract = {The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-ångström-resolution structure of a protofibril formed by a wild-type segment from the β2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29359764,

title = {Structure-based inhibitors of tau aggregation},

author = {P M Seidler and D R Boyer and J A Rodriguez and M R Sawaya and D Cascio and K Murray and T Gonen and D S Eisenberg},

doi = {10.1038/nchem.2889},

year  = {2017},

date = {2017-11-20},

journal = {Nat Chem},

volume = {10},

number = {2},

pages = {170--176},

abstract = {Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29072840,

title = {MicroED Structure of Au₁₄₆(p-MBA)₅₇ at Subatomic Resolution Reveals a Twinned FCC Cluster},

author = {Sandra Vergara and Dylan A Lukes and Michael W Martynowycz and Ulises Santiago and Germán Plascencia-Villa and Simon C Weiss and Jason M de la Cruz and David M Black and Marcos M Alvarez and Xochitl López-Lozano and Christopher O Barnes and Guowu Lin and Hans-Christian Weissker and Robert L Whetten and Tamir Gonen and Miguel Jose Yacaman and Guillermo Calero},

url = {https://cryoem.ucla.edu/wp-content/uploads/2017_vargara.pdf},

doi = {10.1021/acs.jpclett.7b02621},

year  = {2017},

date = {2017-10-26},

journal = {J Phys Chem Lett},

volume = {8},

number = {22},

pages = {5523--5530},

abstract = {Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure, whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28648726,

title = {Taking the measure of MicroED},

author = {Jose A Rodriguez and David S Eisenberg and Tamir Gonen},

url = {https://cryoem.ucla.edu/wp-content/uploads/2017_rodriguez.pdf, Main text},

doi = {10.1016/j.sbi.2017.06.004},

year  = {2017},

date = {2017-10-01},

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

volume = {46},

pages = {79--86},

abstract = {It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}