@article {64873, title = {MicroED data collection with SerialEM.}, journal = {Ultramicroscopy}, volume = {201}, year = {2019}, month = {2019 Mar 19}, 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.

}, issn = {1879-2723}, doi = {10.1016/j.ultramic.2019.03.009}, author = {de la Cruz, M Jason and Martynowycz, Michael W and Hattne, Johan and Gonen, Tamir} } @article {56203, title = {Structural basis for substrate binding and specificity of a sodium-alanine symporter AgcS.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, year = {2019}, month = {2019 Jan 18}, pages = {2086-90}, 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 , 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.

}, issn = {1091-6490}, doi = {10.1073/pnas.1806206116}, author = {Ma, Jinming and Lei, Hsiang-Ting and Reyes, Francis E and Sanchez-Martinez, Silvia and Sarhan, Maen F and Hattne, Johan and Gonen, Tamir} } @article {49180, title = {Analysis of global and site-specific radiation damage in cryo-EM.}, journal = {Structure (London, England : 1993)}, volume = {26}, year = {2018}, month = {2018 Apr 18}, pages = {759-66}, 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.

}, issn = {1878-4186}, doi = {10.1016/j.str.2018.03.021}, author = {Hattne, Johan and Shi, Dan and Glynn, Calina and Zee, Chih-Te and Gallagher-Jones, Marcus and Martynowycz, Michael W and Rodriguez, Jose A and Gonen, Tamir} } @article {49581, title = {MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, year = {2018}, month = {2018 Dec 26}, pages = {13258-63}, 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-{\r A} 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.

}, issn = {1091-6490}, doi = {10.1073/pnas.1806806115}, author = {Purdy, Michael D and Shi, Dan and Chrustowicz, Jakub and Hattne, Johan and Gonen, Tamir and Yeager, Mark} } @article {48156, title = {Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Jan 03}, abstract = {

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP.

}, issn = {2050-084X}, doi = {10.7554/eLife.19273}, author = {Krotee, Pascal and Rodriguez, Jose A and Sawaya, Michael R and Cascio, Duilio and Reyes, Francis E and Shi, Dan and Hattne, Johan and Nannenga, Brent L and Oskarsson, Marie E and Philipp, Stephan and Griner, Sarah and Jiang, Lin and Glabe, Charles G and Westermark, Gunilla T and Gonen, Tamir and Eisenberg, David S} } @article {48265, title = {Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.}, journal = {Nature Methods}, volume = {14}, year = {2017}, month = {2017 Feb 13}, pages = {399-402}, abstract = {

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

}, issn = {1548-7105}, doi = {10.1038/nmeth.4178}, author = {de la Cruz, M Jason and Hattne, Johan and Shi, Dan and Seidler, Paul and Rodriguez, Jose and Reyes, Francis E and Sawaya, Michael R and Cascio, Duilio and Weiss, Simon C and Kim, Sun Kyung and Hinck, Cynthia S and Hinck, Andrew P and Calero, Guillermo and Eisenberg, David and Gonen, Tamir} } @article {48604, title = {A method to minimize condenser lens-induced hysteresis effects in a JEOL JEM-3200FSC microscope to enable stable cryoEM low-dose operations.}, journal = {bioRxiv}, year = {2017}, month = {2017 Jun 21}, abstract = {

Low dose imaging procedures are key for a successful cryoEM experiment (whether by electron cryotomography, single particle analysis, electron crystallography, or MicroED). We present a method to minimize magnetic hysteresis of the condenser lens system in the JEOL JEM-3200FSC transmission electron microscope (TEM) in order to maintain a stable optical axis for the beam path of low-dose imaging. The simple procedure involves independent voltage ramping of the CL1 and CL2 lenses immediately before switching to the focusing and exposure beam settings for data collection.

}, doi = {10.1101/153395}, author = {de la Cruz, Michael Jason and Martynowycz, Michael and Hattne, Johan and Shi, Dan and Gonen, Tamir} } @article {48603, title = {MicroED structures from micrometer thick protein crystals.}, journal = {bioRxiv}, year = {2017}, month = {2017 Jun 20}, abstract = {

Theoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.

}, doi = {10.1101/152504}, author = {Martynowycz, Michael and Glynn, Calina and Miao, Jennifer and de la Cruz, Michael Jason and Hattne, Johan and Shi, Dan and Cascio, Duilio and Rodriguez, Jose and Gonen, Tamir} } @article {47875, title = {Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {113}, year = {2016}, month = {2016 Oct 04}, pages = {11232-6}, abstract = {

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.

}, issn = {1091-6490}, doi = {10.1073/pnas.1606287113}, author = {Sawaya, Michael R and Rodriguez, Jose and Cascio, Duilio and Collazo, Michael J and Shi, Dan and Reyes, Francis E and Hattne, Johan and Gonen, Tamir and Eisenberg, David S} } @article {47730, title = {Atomic resolution structure determination by the cryo-EM method MicroED.}, journal = {Protein Science : a Publication of the Protein Society}, volume = {26}, year = {2016}, month = {2016 Jul 25}, pages = {8-15}, abstract = {

The electron cryo-microscopy (cryoEM) method MicroED has been rapidly developing. In this review we highlight some of the key steps in MicroED from crystal analysis to structure determination. We compare and contrast MicroED and the latest X-ray based diffraction method the X-ray free electron laser (XFEL). Strengths and shortcomings of both MicroED and XFEL are discussed. Finally, all current MicroED structures are tabulated with a view to the future. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.2989}, author = {Liu, Shian and Hattne, Johan and Reyes, Francis E and Sanchez-Martinez, Silvia and de la Cruz, M Jason and Shi, D and Gonen, T} } @article {47422, title = {The collection of MicroED data for macromolecular crystallography.}, journal = {Nature Protocols}, volume = {11}, year = {2016}, month = {2016 May}, pages = {895-904}, abstract = {

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is \~{}10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

}, issn = {1750-2799}, doi = {10.1038/nprot.2016.046}, author = {Shi, Dan and Nannenga, Brent L and de la Cruz, M Jason and Liu, Jinyang and Sawtelle, Steven and Calero, Guillermo and Reyes, Francis E and Hattne, Johan and Gonen, Tamir} } @article {47484, title = {Modeling truncated pixel values of faint reflections in MicroED images.}, journal = {Journal of Applied Crystallography}, volume = {49}, year = {2016}, month = {2016 May 11}, abstract = {

The weak pixel counts surrounding the Bragg spots in a diffraction image are important for establishing a model of the background underneath the peak and estimating the reliability of the integrated intensities. Under certain circumstances, particularly with equipment not optimized for low-intensity measurements, these pixel values may be corrupted by corrections applied to the raw image. This can lead to truncation of low pixel counts, resulting in anomalies in the integrated Bragg intensities, such as systematically higher signal-to-noise ratios. A correction for this effect can be approximated by a three-parameter lognormal distribution fitted to the weakly positive-valued pixels at similar scattering angles. The procedure is validated by the improved\ refinement\ of an atomic model against\ structure factor\ amplitudes derived from corrected micro-electron diffraction (MicroED) images.

}, doi = {10.1107/S1600576716007196}, author = {Hattne, Johan and Shi, Dan and de la Cruz, M Jason and Reyes, Francis E and Gonen, Tamir} } @article {46380, title = {Data Exploration Toolkit for serial diffraction experiments.}, journal = {Acta Crystallographica Section D: Biological Crystallography}, volume = {71}, year = {2015}, month = {2015 Feb}, pages = {352-6}, abstract = {

Ultrafast diffraction at X-ray free-electron lasers (XFELs) has the potential to yield new insights into important biological systems that produce radiation-sensitive crystals. An unavoidable feature of the {\textquoteleft}diffraction before destruction{\textquoteright} nature of these experiments is that images are obtained from many distinct crystals and/or different regions of the same crystal. Combined with other sources of XFEL shot-to-shot variation, this introduces significant heterogeneity into the diffraction data, complicating processing and interpretation. To enable researchers to get the most from their collected data, a toolkit is presented that provides insights into the quality of, and the variation present in, serial crystallography data sets. These tools operate on the unmerged, partial intensity integration results from many individual crystals, and can be used on two levels: firstly to guide the experimental strategy during data collection, and secondly to help users make informed choices during data processing.

}, issn = {1399-0047}, doi = {10.1107/S1399004714025875}, author = {Zeldin, Oliver B and Brewster, Aaron S and Hattne, Johan and Uervirojnangkoorn, Monarin and Lyubimov, Artem Y and Zhou, Qiangjun and Zhao, Minglei and Weis, William I and Sauter, Nicholas K and Brunger, Axel T} } @article {46526, title = {Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals.}, journal = {eLife}, volume = {4}, year = {2015}, month = {2015 Mar 17}, abstract = {

There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.

}, issn = {2050-084X}, doi = {10.7554/eLife.05421}, author = {Uervirojnangkoorn, Monarin and Zeldin, Oliver B and Lyubimov, Artem Y and Hattne, Johan and Brewster, Aaron S and Sauter, Nicholas K and Brunger, Axel T and Weis, William I} } @article {46881, title = {MicroED data collection and processing.}, journal = {Acta Crystallographica Section A: Foundations \& Advances}, volume = {71}, year = {2015}, month = {2015 Jul 01}, pages = {353-60}, abstract = {

MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.

}, issn = {2053-2733}, doi = {10.1107/S2053273315010669}, author = {Hattne, Johan and Reyes, Francis E and Nannenga, Brent L and Shi, Dan and de la Cruz, M Jason and Leslie, Andrew G W and Gonen, Tamir} } @article {47031, title = {Structure of the toxic core of α-synuclein from invisible crystals.}, journal = {Nature}, volume = {525}, year = {2015}, month = {2015 Sep 9}, pages = {486-90}, abstract = {

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 {\r A} resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.

}, issn = {1476-4687}, doi = {10.1038/nature15368}, author = {Rodriguez, Jose A and Ivanova, Magdalena I and Sawaya, Michael R and Cascio, Duilio and Reyes, Francis E and Shi, Dan and Sangwan, Smriti and Guenther, Elizabeth L and Johnson, Lisa M and Zhang, Meng and Jiang, Lin and Arbing, Mark A and Nannenga, Brent L and Hattne, Johan and Whitelegge, Julian and Brewster, Aaron S and Messerschmidt, Marc and Boutet, S{\'e}bastien and Sauter, Nicholas K and Gonen, Tamir and Eisenberg, David S} } @article {45552, title = {Structure of catalase determined by MicroED.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Oct 17}, pages = {e03600}, abstract = {

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 {\r A} resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 {\r A} resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.

}, issn = {2050-084X}, doi = {10.7554/eLife.03600}, author = {Nannenga, Brent L and Shi, Dan and Hattne, Johan and Reyes, Francis E and Gonen, Tamir} }