key: cord-0876030-3779tc8n
authors: Nguyen, Chi; Gonen, Tamir
title: Beyond protein structure determination with MicroED
date: 2020-06-28
journal: Curr Opin Struct Biol
DOI: 10.1016/j.sbi.2020.05.018
sha: 323baeaa44011b42164f6e434adffa9f6fcdb429
doc_id: 876030
cord_uid: 3779tc8n

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.

MicroED [1 ,2 ] has been pushing the limits of cryoEM in determining structures of macromolecular protein assemblies, peptides, and chemical compounds [1 ,3,4 ,5 ]. Prior the technological advancements in detectors [6-9] and software [10] [11] [12] [13] that made the 'cryoEM resolution revolution [14] ' possible, structure determination of biological assemblies, peptides, and chemical compounds has been dominated X-ray crystallography. To date, there are close to 150 000 depositions in the Protein Data Bank (PDB), comprised of roughly 90% X-ray, 8% NMR, and 2% EM structures. The first near atomic resolution structure by cryoEM reported in 2005 [15] set the stage for the highest growth in transmission electron microscopy (TEM) structure deposition that occurred in the last five years.

The field of cryoEM includes at least four major techniques: cryo-electron tomography (cryoET) [16, 17] , single-particleanalysis (SPA) [18] [19] [20] , 2-dimenstional (2D) electron crystallography [21, 22] , and MicroED [1 ,2 ] ( Figure 1 ). All of these cryoEM techniques exploit the advantage that electrons interact orders of magnitude more strongly with materials than X-rays, allowing the application of samples that are not tractable by other methods [23] . While CryoET and SPA use imaging, the crystallographic cryoEM methods of 2D electron crystallography and MicroED also take advantage of electron diffraction. 2D-electron crystallography is typically used for structure determination of moleucles in 2D arrays, which traditionally have been of membrane proteins that are crystallized within the native environment of the lipid bilayer [22] . In contrast, MicroED uses 3D crystals and data collection by continuous rotation to yield structures of a wide range of samples including soluble and membrane proteins, peptides, small organic and inorganic molecules, semi-conductors, and natural-products [5 ] .

During MicroED experiments, crystals are harvested and prepared in a number of ways for embedment on EM grids (discussed below) [1 ] (Figure 2 ). Biological samples, which are more sensitive to radiation, are typically vitrified to protect from radiation damage and to withstand the high-vacuum within the electron microscope [1 ,27] . Once well-diffracting crystals are detected, MicroED datasets are collected by exposure of the sample to an electron beam in diffraction mode during continuous rotation of the stage [2 ] ( Figure 2 ). MicroED data are then collected on a fast camera as a movie, where each frame contains a diffraction pattern that represents a wedge of the reciprocal space [27] . Because continuous rotation for MicroED is analogous to the rotation method in X-ray crystallography, the data collected can be directly processed by existing standard X-ray crystallography software such as Mosflm [28] , XDS [29] , DIALS [30] , SHELX [31] and HKL2000 [32] (Figure 2 ).

After data-processing, the phases are determined and structures are built using the electron density maps [1 ]. Given its wide-application and ability to extract structural information from nanocrystals, often a billionth the volume of those needed for X-ray crystallography, there are a growing number of structures determined by MicroED. Since its inception in 2013, there are close to 100 PDB entries produced by MicroED, with the highest growth in the just the past two years. To date, several laboratories have published MicroED studies and the number of practitioners are growing. Still, there are several challenges that lay ahead for MicroED including additional methodologies for sample preparation and technological developments for data collection that currently limit widespread usage of the technique. Below we discuss the most recent developments and strategies to expand the use of the MicroED.

Formation of large crystals continue to be the most challenging and time-consuming step for X-ray crystallography, especially for membrane proteins and protein complexes [33] . The small crystals that are typically formed by membrane proteins and protein complexes can often diffract electrons using very low exposures to minimize radiation damage (0.01 e À /Å2) [1 ]. One of the earliest membrane protein structures determined by The four major modalities of cryoEM. From left to right. A model of the HeLa cell nuclear periphery by Cryo-ET (Reprint from Ref. [24] with permission from AAAS). Model of COVID-19 spike protein by SPA (Reprint from Ref. [25] with permission from AAAS). The structure of bacteriorhodopsin determined by electron crystallography (Reprint from Ref. [21] ). The structure of catalase determined by MicroED (Reprint from Ref. [26] ). MicroED workflow. From left to right. Crystals are grown, harvested, and placed directly on EM grids. Manual or automated screening to assess for electron diffraction of the crystals. When a well-diffracting crystal is identified, a dataset is collected by continuous rotation. The diffraction dataset is then processed using standard crystallography software to allow model building and structure refinement. + ATPase, the structure of NaK by MicroED allowed generation of Coulomb potential maps to unambiguously place Na + within the channel and to visualize a new transient state of the channel [35 ] . The heterodimeric complex between TGF-bm and TbRII plays essential roles in the adaptive immune response and maintenance of the extracellular matrix [38] . Unlike Ca 2+ ATPase and NaK, which formed nanocrystals, the structure of TGFbm:TbRII was obtained from fragmentation of large, imperfect crystals (discussed below) [36]. Compared with the imperfect, large parent crystals, this approach led to better MicroED data from well-ordered microcrystal fragments that ultimately yielded atomic-resolution structures [36] . This study expanded the application of MircoED to include a wider range of crystal sizes.

MicroED has already made important contributions to drug discovery by determining structures of protein-drug complexes and supra-resolution of small-molecules and Microcrystal electron diffraction Nguyen and Gonen 3 (Figure 3c ). The HIV-GAG-bevirimat complex provided important information about the antiviral drug mechanism and was the first demonstration of drug discovery using MicroED.

MicroED was originally intended for studying protein assemblies [26] , however, it was rapidly recognized that this technique is a powerful tool for the characterization of small-molecules and natural-products. In 2016, the structure of the sodium channel blocker carbamezapine was determined to $1 Å resolution [40] (Figure 3d ). In 2018, a method for small-molecule sample preparation using a "powder to structure" pipeline was described for carba- , an antiviral drug for the treatment of the common cold and the flu [43] , and a recent example of the non-fulleren acceptor (NFA) semi-conductive material ITIC-Th (Figure 3d ).

MicroED has also proven its usefulness for the structure characterization of several natural-products that have previously been challenging or, in some cases, impossible to determine by other techniques. Unlike their synthetic small-molecules counterparts, biosynthesized naturalproducts are typically larger, structurally dynamic, obtained in small amounts, and difficult to crystallize, posing considerable challenges for X-ray studies. Even when natural-products form lattices, these crystals are often too small and are not useful for X-ray diffraction [44 ,45 ] . Brucine is an alkaloid toxin currently being tested for its anticancer properties [46] (Figure 3e ). The MicroED structure of brucine at 0.9 Å resolution allowed for definitive assignment of its two chiral centers, key for understanding its toxicity and anticancer properties [4 ] (Figure 3) . Brucine, while large compared to small-molecules, is relatively small compared to amino-acid derived natural-products called ribosomally synthesized and posttranslationally modified peptides (RiPPs), including 3-thiaGlu [44 ] and thiostreptin [47] (Figure 3e ). Glutamylated thiols, similar to the peptide modification on 3-thiaGlu, have been shown to block jasmonate and ethylene signaling pathways [48] . Thiostreptin is an antibiotic currently used in veterinary medicine [47] (Figure 3e ). When efforts failed by X-ray crystallography, MicroED readily provided a 0.9 Å resolution of the 3-thiaGlu peptide (PDB 6PO6) [44 ] . While thiostreptin has been studied by NMR [44 ] and X-ray crystallography [49] previously, the ease of its characterization speaks to the robustness of MicroED for structure determination of large, flexible natural-products (Figure 3e ). Like the difficulties encountered for 3-thiaGlu, the structures of 3-substituted oxindole derivatives (Figure 3e ), that contain a new stereocenter at the g carbon installed by an enzyme through directed-evolution, was only solved with the application of MicroED [45 ] . These recent studies demonstrate that MicoED provides an additional strategy for more complete analysis of absolute configuration based on internal markers [44 ,45 ] .

Electrons interact much more strongly with material than X-ray [23] . This phenomenon, however, results in high absorption and, thus, electrons can only penetrate very thin materials. Crystals that are greater than 500 nm in thickness must be thinned before fragmentation has been successful for determining protein structures from large crystals of lysozyme, TGF-bm TbRII, xylanase, thaumatin, trypsin, proteinase K, thermolysin, and a segment of the protein tau [36] . Moving forward, the most current and promising technique for trimming large crystals for MicroED is FIB milling [50, 51, 52 ] . During FIB milling, a crystal is repeatedly exposed a gallium beam to trim away the surrounding materials and generate lamellas with controllable thicknesses. As proof of principle, the structures of several proteins, including lysosome and proteinase K, have been determined by FIB mill and MicroED [50,51,52 ,53,54] ( Figure 4) . Currently, FIB milling crystals is a relatively slow process but,even then, about ten crystal lamellas can be prepared per day.

Radiation damage in structural studies continue to be a major challenge leading to poor processing statistics and map quality [55, 56] . When electrons penetrate materials, they deposit energy that can deteriorate the samples, a process referred to as radiation damage. Radiation damage can be categorized into two forms: global and Microcrystal electron diffraction Nguyen and Gonen 5 site-specific. Global radiation damage typically results in the disruption of the crystal lattice which can be detected during data-processing when decreases in overall diffraction intensities and increases in B-factors are observed [57, 58] . On the other hand, site-specific radiation damage is not uniform, is not typically detected during dataprocessing, and observable only during examination of the real-space map [59] . The degree of radiation damage depends, among other things, on the content of the sample, the surrounding solution, and is proportional to the amount of energy used during diffraction studies. For MicroED, site-specific radiation damage has been illustrated to occur on specific amino-acids including cysteines, glutamates, and aspartic acids [60 ] . To curb the effects of radiation damage, samples are often vitrified [61] . However, even the combination of vitrification and exposure to extremely low doses of electrons (0.01 e À /Å2/ s) during MicroED experiments can still lead to detectable radiation damage [60 ] .

TEMs for cryo-EM studies are typically equipped with highly-sensitive direct-electron detectors designed for imaging [9, 14, 62] . These highly sensitive cameras, however, have not been used extensively for MicroED because of concerns of damage to the sensors. As such, MicroED data are typically collected on indirect-electron detectors such as the complementary metal oxide semiconductor (CMOS)-based CetaD and TVIPS TemCam-F416 cameras. This strategy, however, limits the availability of MicroED because most facility TEMs are typically outfitted with top-of-the-line direct-electron detectors for imaging and not CMOS cameras. Recently, the Falcon III direct-electron camera was tested for MicroED data collection [63 ] . This study demonstrates that MicroED data collected at lower electron exposure, to avoid camera damage of Falcon III, lead to greater mean completeness relative to CMOS detectors and to higher quality maps. As proof of principle, examination of the maps of proteinase K from data collected on the Falcon III camera preserved the disulfide bonds,which are highly susceptible to radiation damage [63 ] ( Figure 5a) . A similar approach has been used recently with a Gatan K2 direct-electron detector in counting mode with an exposure 25 times less than for the Falcon III detector and a seemingly damage-free structure of Proteinase K has been determined.

Outrunning radiation damage using the direct electron detector Falcon III has been instrumental in determining the structures of samples that are highly susceptible to damage [44 ,45 ] and we believe that many more such examples would be forthcoming. Radiation damage was recently exploited to establish a pipeline for phasing MicroED data [64 ] (Figure 5b) . A low-damage followed by a high-damage data sets were taken from the same crystal. A difference Patterson was calculated and allowed for generation of initial phases. Following cycles of manual model building and refinement, the structure of a peptide was determined (Figure 5b ). This study demonstrates the ability to extract meaningful phase information using radiation damage in MicroED.

MicroED is proving to be an important new tool in structural biology not only in determining structures of proteins but also of peptides, small organic and inorganic molecules, and natural-products. Continuous rotation MicroED is paradigm shifting because it has proven to be a robust, fast, and efficient method for structure determination of small molecules and natural products, even without crystallization and directly from mixtures [4 ] . To our knowledge, no other structural biology method is capable of determining atomic resolution structures directly from mixtures, making MicroED a useful and powerful tool for an array of problems that are yet to be explored. Moving forward, the application of FIBmilling and fast-cameras will certainly expand MicroED for structure determination of varying types of samples with a wide-range of crystal sizes and to facilitate time resolved studies. These advancements could ultimately be applied to establish automated pipelines that mirror those for X-ray crystallography to facilitate obtaining MicroED structures. Early examples of automation in MicroED has already reported in which several hundred data sets could be collected overnight to demonstrate similar throughput as synchrotrons [65] .

Nothing declared.

Jones CG et al.: The CryoEM method MicroED as a powerful tool for small molecule structure determination. ACS Cent Sci 2018, 4:1587-1592 After the successful application of MicroED for proteins, this techniques was applied for structure determination of small-molecules and naturalproducts. These studies tout a 'powder to structure' pipeline where structures of small-molecules and natural-products were obtained minutes after sample preparation and MicroED data collection. Further, this work illustrates the precision of MicroED in obtaining structures from polymorphic mixtures of small-molecules and natural-products. 

Achieving better-than-3-Å resolution by single-particle cryo-EM at 200 keV

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

K3 -a first look at the new direct electron detection camera from Gatan company

SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM

New tools for automated high-resolution cryo-EM structure determination in RELION-3

Real-time cryo-electron microscopy data preprocessing with Warp

CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

The resolution revolution. Science (80-.)

Lipid-protein interactions in double-layered two-dimensional AQP0 crystals

Magnetosomes are cell membrane invaginations organized by the actin-like protein MamK

Electron cryotomography of the E. coli pyruvate and 2-oxoglutarate dehydrogenase complexes

Cryo-electron microscopy of vitrified specimens

Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning

Advances in structural and functional analysis of membrane proteins by electron crystallography

Model for the structure of bacteriorhodopsin based on high-resolution electron cryomicroscopy

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules

Visualizing the molecular sociology at the HeLa cell nuclear periphery

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science (80-.) 2020

Structure of catalase determined by MicroED

The collection of microED data

iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM

Diffraction-geometry refinement in the DIALS framework

Structure solution by iterative peaklist optimization and tangent expansion in space group P1

Evaluation of the efficacy of a combined formulation (GrippostadJ-C) in the therapy of symptoms of common cold: a randomized, double-blind, multicenter trial

Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products

RiPP with a novel peptide modification with biological activities, failed to generate a structure by Xray crystallography. MicroED achieved a 0.9 Å resolution of 3-thiaglu to allow assignment of the absolute configuration of its stereocenters, providing important insights into the mechanism for the installation of this unique modification

Tailoring tryptophan synthase TrpB for selective quaternary carbon bond formation

Crystals of 3-substituted oxindole derivatives, which contain a new stereocenter at the g carbon that were installed by the TrpB enzyme through directed-evolution, failed to generate structures by X-ray crystallography. MicroED achieved structure determination of several of these 3-substituted oxindole derivatives at supra-resolution

Antitumor effects of brucine immuno-nanoparticles on hepatocellular carcinoma in vivo

Antimicrobial activity of thiostrepton: tube dilution studies

A: Chemical and metabolic aspects of antimetabolite toxins produced by Pseudomonas syringae pathovars

The structure of thiostrepton

Collection of continuous rotation MicroED data from ion beam-milled crystals of any size

Machining protein microcrystals for structure determination by electron diffraction

e2 FIB milling has been establishes as a tool to expand MicroED for structure determination of large crystals. Here, these studies applied an additional step called 'polishing' to improve the MicroED diffraction quality of crystals from FIB milling

In situ protein micro-crystal fabrication by cryo-FIB for electron diffraction

Using focus ion beam to prepare crystal lamella for electron diffraction

When X-rays modify the protein structure: radiation damage at work

Radiation damage in electron cryomicroscopy

Quantifying X-ray radiation damage in protein crystals at cryogenic temperatures

Unit-cell volume change as a metric of radiation damage in crystals of macromolecules

Specific chemical and structural damage to proteins produced by synchrotron radiation

Analysis of global and site-specific radiation damage in Cryo-EM

The first comprehensive study to decipher the effects of radiation damage during MicroED experiments with implications to all cryoEM studies. These studies found both global and site-specific radiation damage during MicroED studies

Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate

Single-particle cryo-EM data acquisition by using direct electron detection camera

MicroED with the Falcon III direct electron detector

Experimental phasing of MicroED data using radiation damage

This study demonstrates the use of radiation damage for ab initio phasing with MicroED

MicroED data collection with SerialEM

We would like to thank the Gonen laboratory and all collaborators who worked with us on MicroED applications. The Gonen lab is funded by the Howard Hughes Medical Institute and the National Institutes of Health P41-GM136508.

Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.Shi D, Nannenga BL, Iadanza MG, Gonen T: Three-dimensional electron crystallography of protein microcrystals. eLife 2013, 2:1-17 These studies mark the development and launch of MicroED from Janelia Research Campus for protein structure determination. Using lysozyme as a model system, the first MicroED structure of this protein was solved to 2.9 Å resolution.

Nannenga BL, Shi D, Leslie AGW, Gonen T: High-resolution structure determination by continuous-rotation data collection in MicroEDED. Nat Methods 2014, 11:927-930 Important accompanying studies to the launch of MicroED in 2013 that illustrates continuous-rotation data collection. This work demonstrates that continuous-data collection for MicroED leads to better processing statistics, which can be achieved using already existing X-ray crystallography software.