The cryo-EM method microcrystal electron diffraction (MicroED)

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.

Main

Cryo-EM encompasses a variety of techniques that use a transmission electron microscope (TEM) to determine the structures of frozen-hydrated beam-sensitive samples (Fig. 1). Cryo-EM techniques can make use of two modes of data collection: those that use images of biological samples (purified biomolecules or whole cells), and crystallographic approaches based on diffraction. In single-particle cryo-EM, images of individual biomolecules are collected, and then image processing is carried out to generate 3D reconstructions of those biomolecules¹. In cryo-electron tomography, images of biomolecules within their native cellular environment are collected²; however, although important biological insights can be gained from tomography, the resolution in this method is substantially lower than that of all other cryo-EM methods.

Fig. 1: Methods in the field of cryo-EM.

The diverse imaging-based and diffraction-based techniques used in cryo-EM can provide structural information from a wide range of samples. Images acquired with the different techniques are shown here, depicting the synaptosome⁹⁴ (electron tomography), a 2.2-Å reconstruction of β-galactosidase⁹⁵ (single-particle reconstruction), a 1.9-Å structure of aquaporin-0¹⁰ (2D electron crystallography) and a 1.0-Å structure of the NNQQNY Sup35 prion fragment⁴⁹ (MicroED). Adapted with permission from ref. ⁹⁴, Oxford University Press.

The crystallographic cryo-EM techniques of 2D electron crystallography^3,4 and MicroED^5,6 both use crystalline arrays of material. However, because crystals are 2D in electron crystallography and 3D in MicroED, the data collection and processing differ substantially between the two techniques, which renders them distinct branches of cryo-EM modalities. For example, a key difference is that in 2D electron crystallography, the orientation of the 2D array can be determined from a single diffraction pattern because the one crystallographic axis is always parallel to the beam in every crystal. In contrast, it is rare for any crystallographic axis to be parallel to the beam in 3D crystals. Therefore, with MicroED, the user must collect a wedge of data in reciprocal space^7,8 rather than a single diffraction pattern, and this in turn is processed differently than the data for 2D crystals. 2D electron crystallography has a long and storied history; many of the first high-resolution cryo-EM structures were determined from 2D crystals^{9,10,11,12,13,14,15}. Electron diffraction also has been used to investigate thin 3D protein crystals for several decades^{16,17,18,19,20,21}. Despite the early observations that 3D protein microcrystals can produce high-resolution diffraction data, the use of 3D microcrystals for structure determination by cryo-EM was not achieved until 2013, with the development of MicroED⁷.

MicroED takes advantage of highly sensitive modern cryo-EM detectors to determine protein structures from nanocrystals only about ten layers thick, as we demonstrated with the first complete high-resolution electron-diffraction structure of lysozyme⁷. The initial ‘still-diffraction’ MicroED data-collection procedure⁷ facilitated the correct indexing, data processing and structure determination of lysozyme to 2.9-Å resolution with specialized software²². For this, the crystal was tilted at defined angles within the cryo-TEM while diffraction was recorded.

In early 2014, we improved MicroED data collection by introducing ‘continuous rotation’, whereby the crystal is continuously rotated within the electron beam as data are recorded on a high-speed detector as a movie⁸ (Fig. 2). Because continuous rotation is analogous to the rotation method used in X-ray crystallography, data collected by this method can be processed with well-established software that was developed for X-ray crystallography. Continuous rotation therefore improved the quality of the raw data by increasing the sampling of reciprocal space, reducing dynamic scattering (Box 1) and improving data processing, the combination of which ultimately yielded improved final structures. The structure of lysozyme determined by continuous rotation was initially reported at 2.5 Å with significantly improved data-processing statistics⁸. With further data-processing improvements^23,24, this structure was improved to 1.8-Å resolution (Fig. 3).

Fig. 2: MicroED overview.

From left to right: MicroED data are collected as movies while the stage of the cryo-electron microscope is continuously rotated. This produces a series of high-resolution diffraction patterns that can be processed to produce high-resolution structures directly from microcrystals. Here the structure of the nonselective ion channel NaK is illustrated⁹⁶.

Fig. 3: Improvements in MicroED data quality.

Continual development of MicroED has led to steady improvements in the quality of structures obtained. This can be seen by the increases in resolution that are possible from similar lysosome microcrystals. Density maps (2F_o–F_c) in gray are contoured at 1.5σ for all structures shown. Panels from left to right correspond to refs. ^7,8 and ²⁴, respectively.

Continuous-rotation data collection represents the standard data-collection approach by which all MicroED structures (except for the very first lysozyme structure from 2013) have been determined to date. Thus far, approximately 40 protein, peptide and small-molecule structures have been determined by MicroED since 2013; several of these are novel structures that resisted other methods (Fig. 4, Table 1 and described in detail below). Centers around the globe are adopting the technology, and several structures have been published by others in the past couple of years^25,26.

Fig. 4: Examples of novel structures determined by MicroED.

a, The NACore fragment from α-synuclein was determined to 1.4-Å resolution. b, Gag–bevirimat MicroED structures (side and top views shown in surface and ribbon representation, respectively), in which the location of the bevirimat (arrows) and its interactions with the Gag complex can be identified. c, 2F_o–F_c density map (contoured at 2.0σ) from MicroED data of Au₁₄₆(p-MBA)₅₇, in which rows of ligand-stabilized gold atoms can be clearly seen. The modeled atoms are shown in the inset.

Table 1 Examples of MicroED structures

MicroED is different than other electron-diffraction techniques that have been reported over the years, such as automated diffraction tomography (ADT)²⁷ and rotation electron diffraction (RED)²⁸. ADT uses discrete tilting of the goniometer together with beam precession²⁷, whereas RED uses discrete goniometer tilts combined with beam tilting to sample reciprocal space, and both techniques require specialized software to control data collection and process the data that are collected. These techniques were developed and used on a variety of inorganic and organic samples from materials science^{29,30,31,32,33,34,35,36}, which can generally tolerate a higher electron dose than biological materials. In contrast, MicroED was developed for structure determination of proteins and radiation-sensitive biological material. MicroED uses very low dose rates (~0.01 e^–/Ų/s) and relies only on stage rotation to collect data, in a manner analogous to the rotation method in X-ray crystallography³⁷, which facilitates data processing by standard crystallographic programs³⁸. Although MicroED was initially developed for biological material, it has now also been used successfully on small molecules^39,40 and inorganic material⁴¹.

X-ray crystallography methods and MicroED

The growth of large and well-ordered crystals represents the largest bottleneck for structure determination by crystallographic approaches, especially for difficult samples such as membrane proteins and protein complexes⁴². Therefore, new methods that are capable of producing diffraction data from smaller and smaller crystals are of great interest to the structural biology community. Additionally, it has been shown that for some systems, small crystals are better ordered and produce higher-quality diffraction data than larger crystals. One approach relies on the more intense and smaller microfocus beamlines at synchrotron facilities, and thus allows the use of samples smaller than tens of micrometers⁴³; however, the high flux of the microfocus beam can cause issues with radiation damage from these small crystals⁴⁴. X-ray free-electron lasers (XFELs) use a much brighter beam than traditional synchrotrons, and as a result, meaningful data can be obtained from crystals as small as ~1 μm; crystals smaller than this are usually destroyed after only a single exposure⁴⁵. However, because the pulse is so short—in the femtosecond range—the diffraction data are collected before the results of damage are seen. In this method, each crystal produces only one image; therefore, XFEL data usually consist of partially recorded reflections, and data from thousands of crystals must be collected and merged to yield a complete dataset. Although XFELs are producing exciting and impactful results⁴⁶, the hardware required is extremely expensive to build, maintain and use, which limits the accessibility of this technique.

Relative to X-rays, electrons deposit considerably less damaging energy into a crystal per useful elastic scattering event⁴⁷. For this reason, electron-diffraction data can be collected from extremely small micro- and nanocrystals at an ultra-low dose that allows many exposures from a single crystal. MicroED data collection, described in more detail below, typically allows for the collection of up to a 140° wedge of data from a single crystal, and the data recorded are of mostly fully measured intensities. The measurement of full reflections leads to improved data processing and high-quality final data relative to results obtained with only partial reflections⁸. If the crystal orientation and symmetry are favorable, data collection from a single crystal can be enough to determine a high-quality structure^8,48. The ability to determine structures from a single crystal or from the merging of a small number of crystals greatly enhances the throughput of MicroED relative to that of other multi-crystal X-ray techniques. In cases where crystals diffract well in cryo-EM, the entire process of data collection and structure solution can be performed in under an hour. Moreover, the hardware required for a MicroED experiment is orders of magnitude less expensive than that for XFELs, and the cost per hour of operation and amount of sample needed are likewise orders of magnitude lower. The resolutions obtained from several MicroED samples^{39,41,49,50,51,52} have been higher than those achieved with XFELs, which shows that MicroED is a competitive method in microcrystallography.

Box 1 About dynamical scattering and crystal thickness

The effects of dynamical scattering, also known as multiple scattering, have been a topic of much investigation and debate in all areas of electron diffraction. Structure-determination efforts generally assume that electrons are diffracted by the crystal a single time, in what is known as kinematic scattering; however, as crystals become thicker, the probability that electrons will be diffracted multiple times within the crystal increases until the intensities of the reflections are so severely redistributed that the diffraction patterns can no longer be reliably used for structure interpretation. This is known as dynamical scattering. Despite reports in 1975 that studies of thin 3D crystals of catalase had shown that kinematic scattering could be assumed for protein crystals in electron diffraction¹⁷, it was alternatively commonly assumed that dynamical scattering would be much too severe for protein crystal diffraction data, which essentially halted progress in the field for decades. Moreover, simulations indicated that when crystals thicker than only ~50–100 nm are used, dynamical scattering will be so severe that the underlying relationship between the structure factors and the recorded intensity will be completely lost⁹⁷. The initial proof-of-concept MicroED paper, published in 2013, confirmed that multiple scattering does not preclude structure determination from thin 3D crystals, which in this case were approximately 200-nm-thick lysozyme crystals. Analysis of the systematic absences of the lysozyme diffraction data determined that data derived from still diffraction patterns contained approximately 5% error that was attributed to dynamical effects⁷.

Precession electron diffraction is a method used in materials science to reduce the effect of dynamical scattering by pivoting the electron beam using the coils of the TEM during exposure⁹⁸. In this approach, dynamical scattering is diminished because as the beam is rotated, the secondary scattering is averaged, leading to an ensemble of near-kinematical recorded diffraction intensities⁹⁹. Precession electron diffraction has been successfully used to determine several structures in materials science from inorganic samples that are radiation hardy, through the ADT and RED methods¹⁰⁰. Continuous-rotation MicroED is similar to precession, except rather than the beam being rotated, the crystal is rotated in the beam during exposure. With continuous-rotation MicroED, we demonstrated a significant reduction in the error associated with multiple scattering in lysozyme (estimated at 2.5% for the same lysozyme crystals that without continuous rotation showed a 5% error) and an improvement in the final integrated data obtained by MicroED⁸.

The most convincing evidence for the negligible effects of dynamical scattering on continuous-rotation MicroED data came from the ab initio structure determination of the yeast prion protein Sup35⁴⁹. These crystals were estimated to be 300–400 nm thick and produced diffraction data that extended to 1 Å. This level of resolution allowed the phasing of the data by direct methods. Because direct methods use only the measured intensities, the successful ab initio phasing and structure determination of these samples demonstrated that dynamic scattering represents a minor component of the error observed in MicroED data. Consistent with these results, Friedel-pair analysis of the data indicated no detectable difference between the reflection pairs, thus strongly demonstrating the validity of the kinematic scattering assumption for continuous-rotation MicroED. Taken together, these results, and those from several other structures that followed, clearly show that highly accurate data can be collected by continuous-rotation MicroED from crystals with a range of sub-micrometer thicknesses.

MicroED data collection and analysis

The workflow for MicroED has elements similar to both cryo-EM and X-ray crystallography. The electron microscopist will be familiar with the grid preparation and operation of the cryo-electron microscope, while X-ray crystallographers will be accustomed to the procedures of crystal growth, data processing and refinement involved in MicroED. Data-collection methods are also similar to the rotation method used in X-ray crystallography. In-depth protocols on MicroED data collection and processing have been published previously^37,38, and readers are encouraged to consult them for guidance on using MicroED.

As with any crystallographic technique, the initial step of MicroED is the identification of diffraction-quality crystals and sample preparation for irradiation. Light microscopes can be used to identify conditions that produce nanocrystals, but with difficulty. This is because in certain cases the nanocrystals used for MicroED may be ‘invisible’ when viewed by a conventional light microscope. Improved methods for crystal identification involve the use of UV fluorescence (Fig. 5) or, in some cases, second-order nonlinear optical imaging of chiral crystals⁵³, both of which are capable of identifying potential crystals on the nanometer scale. If conditions are identified with potential microcrystals, negative-stain electron microscopy can be used to confirm the presence and quality of the microcrystals present in the drop^54,55,56. However, it is important to note that the negative stain might be incompatible with certain crystallization buffers or lead to the deterioration of crystal quality. Thus, visualization of high-quality microcrystals by negative stain indicates that MicroED studies are likely to be successful, but the absence of well-ordered crystals does not necessarily indicate that the quality of the crystals is poor. A useful alternative is to freeze a crystal slurry on a sample loop and use powder-type X-ray diffraction at a synchrotron. No coherent lattice will be observed, but if diffraction rings are seen, the presence of protein nanocrystals in the solution and the potential resolution can be confirmed. Additionally, it has been shown that larger, imperfect crystals can be broken down by a variety of methods (for example, vortexing, sonication or vigorous pipetting) to produce crystal fragments of suitable size for MicroED²⁴.

Fig. 5: Crystal identification and sample preparation for MicroED.

a, Frequently, the identification of microcrystals in drops that appear to have cloudy precipitates is difficult by visible light (left); however, when the drops are imaged using UV fluorescence, the microcrystals are clearly seen as sharp glowing spots (right). b, UV fluorescence can also be used to visualize the presence of microcrystals deposited on an electron microscopy grid during sample preparation. Scale bars, 500 μm.

Once nanocrystals suitable for MicroED have been identified, samples are prepared by vitrification methods analogous to those used for other cryo-EM techniques. As with all cryo-EM modalities, grid preparation for imaging is difficult and empirical. Grid preparation and optimization can often be the most delicate and time-consuming step of a cryo-EM experiment, especially for sensitive samples, and generalized procedures for difficult targets (e.g., membrane proteins) are still an area of development. Samples must be blotted until they are thin enough for the electron beam to penetrate, yet the sample must also not be too disturbed during the process. Blotting times and, often, the use of low-viscosity cryo-protectants (e.g., 2-methyl-2,4-pentanediol) must be optimized for MicroED experiments. Generally, samples are applied to a holey carbon grid that has been glow-discharged on both sides. The grids are then blotted and vitrified in liquid ethane with either a manual or an automated plunge-freezing device. Even though holey carbon grids are used, unlike for single-particle cryo-EM, it is not necessary that the crystals be in the holes. Indeed, most MicroED data are collected from crystals on the carbon surface rather than in the holes. For MicroED, these carbon grids are used to help with efficient blotting rather than imaging in the diffraction experiments. After vitrification, cryo-EM grids may be stored indefinitely under liquid nitrogen until the user is ready to perform MicroED.

An alternative, promising method for preparing appropriate crystalline specimens for MicroED is the use of a focused ion beam (FIB) under cryogenic conditions (cryo-FIB) to mill thicker specimens down to thicknesses in the hundreds of nanometers for diffraction data collection. For this approach, nanocrystals are placed directly on the electron microscope grid without blotting, the grids are plunged into ethane, and crystals are milled with the FIB to the desired thickness (Fig. 6). After milling, the grids are transferred into a TEM for MicroED data collection. Because cryo-FIB milling can help alleviate the challenges associated with the use of viscous samples and with sample damage during blotting, we predict that once the availability of cryo-FIB systems becomes more widespread, this will be a standard method by which samples are prepared for MicroED⁵⁷.

Fig. 6: Cryo-FIB milling of a thick crystal.

a, Sample preparation for MicroED: a cryo-FIB was used to mill down thick crystals to a few hundred nanometers. Red arrows indicate the unmilled crystal (left) and the thin lamella after milling (right). b, After cryo-FIB milling, the grid would be loaded into the TEM, and diffraction data would be collected from the thin lamella. c,d, The final structure of proteinase K. Shown are a representative region of the model and density map (c) and a ribbon representation of the entire structure (d).

The cryo-TEM must be set up appropriately for MicroED data collection. Most modern cryo-TEMs equipped with quality detectors for diffraction (described more below) are capable of collecting MicroED data, and as with all other cryo-EM techniques, the cryo-TEM must be well aligned and calibrated. A field emission gun (FEG) as an electron source is highly desirable for increased beam coherence, together with a well-calibrated sample stage. An energy filter helps by cutting out the inelastic scattering, which effectively reduces noise and helps to identify faint reflections from weakly diffracting crystals. As a result, the energy filter is important not only for data collection but also for screening (Fig. 7).

Fig. 7: Comparison of proteinase K data collected with and without an energy filter.

The zero-loss data collected on a cryo-TEM equipped with an in-column energy filter show much less diffuse scattering at lower resolutions.

Microscope setup for MicroED data collection has been described previously³⁷. The next step is to search the grid at low magnification to identify the location of suitable crystals. High-quality crystals show clearly defined edges and are well separated from other crystals on the grid. If a promising crystal is identified, a test diffraction pattern should be collected to determine the diffraction quality of that crystal: high-quality crystals will diffract to high resolution and show well-separated reflections. The identification of well-diffracting crystals and troubleshooting have been described before in detail⁵⁸. We recommend that new MicroED users become familiar with the method and what is considered high-quality diffraction data by testing standard samples. Common protein samples that have been used to benchmark MicroED instruments and protocols are lysozyme, catalase and proteinase K^8,23,48. Of these samples, we prefer to use proteinase K, as a higher number of crystals on the grid show high-quality diffraction.

A diffraction dataset is next collected from crystals deemed high quality. The first step is to set the eucentric height accurately to ensure that the crystal remains in the beam as the sample is tilted throughout the rotation range of the stage. For a well-aligned sample stage, the crystal can remain in the beam over an entire 140° rotation (±70°). For the collection of continuous rotation data, the stage is smoothly and unidirectionally rotated at a constant rate while the detector constantly collects the diffraction data as a movie. The detector readout system must be fast enough that the delay between write-out of the frames of the movie is negligible. The camera used for most MicroED structures is the CMOS (complementary metal-oxide semiconductor)-based TVIPS F416, but other CMOS detectors with fast readouts, such as the Thermo Fisher CetaD, Gatan OneView and TVIPS XF416, are also suitable and have been used. These cameras are widely available and are much less expensive than the direct electron detectors used for imaging.

After MicroED data have been collected, the data are integrated, scaled and merged using software developed for X-ray crystallography, which makes the process relatively straightforward for users with crystallography experience. We recommend that users carry out MicroED data integration with whatever program they feel most comfortable. The following suites have been tested and used: MOSFLM^59,60, XDS⁶¹, HKL2000⁶² and DIALS^63,64. It should be noted that the initial indexing of the crystal is different in MicroED than in X-ray crystallography. Because the de Broglie wavelength of the electrons is significantly shorter than the wavelength for X-rays (for example, 0.025 Å for electrons in a 200-kV TEM versus 1.54 Å for X-rays from a copper source), the Ewald sphere is essentially flat at the resolutions typical for protein crystallography. This produces diffraction patterns that sample planes of reciprocal space, with very little information about the third dimension in a single image. Therefore, MicroED requires multiple diffraction patterns that span a tilt range of at least 20° to obtain the 3D information necessary to index and determine the crystal orientation successfully without any prior knowledge. After data integration, merging and scaling, standard crystallographic software suites such as CCP4⁶⁵ and PHENIX⁶⁶ are used to phase the data and refine the model. Aside from high-resolution peptides, small molecules and metals, which can be phased by direct methods, all MicroED structures have been phased thus far by molecular replacement. Refinement, typically performed with REFMAC⁶⁷ or phenix.refine⁶⁶, is done with the same programs and procedures used for X-ray crystallographic data, with the exception of the use of electron scattering factors rather than X-ray scattering factors. The ability to use electron scattering factors in REFMAC and phenix.refine is already a feature that has been added by the program developers.

MicroED structure-determination highlights

Since its inception, MicroED has been used to determine several structures that were previously unsolved despite major effort, such as the structures of fragments derived from amyloidogenic proteins⁶. The hallmark of amyloid diseases is the formation of amyloid fibrils, which are composed of proteins (for example, amyloid-β in Alzheimer’s and α-synuclein in Parkinson’s) that have adopted an aggregation-prone β-sheet morphology. Determining the structure of these fibrils and their oligomeric precursors is key to the understanding of the molecular mechanisms of amyloid disease progression and the design of novel therapeutics. Peptide fragments from amyloidogenic proteins composed of four to seven residues could produce large crystals suitable for analysis by X-ray crystallography^68,69,70. However, as the number of residues increases, the size of the crystals is reduced and limited, presumably because of the increased strain associated with the twisting of the β-sheets. The 11-residue fragment from α-synuclein, which is called the NACore, yielded crystals that were only a few nanometers in size and resisted structure determination for over a decade. Multiple researchers over the years were unsuccessful at growing these nanocrystals to a size that would allow X-ray diffraction. Milliliters of these nanocrystals were sent for analysis at XFELs, but even XFELs failed to deliver a structure. These α-synuclein nanocrystals were the first novel samples investigated by MicroED, which yielded a 1.4-Å dataset and a fully refined structure in just a few days⁷¹ (Fig. 4a). Since this first structure of the NACcore was published, MicroED has been used to solve a number of other amyloidogenic structures with crystals too small for other techniques, including those from islet amyloid peptide, amyloid-β, Tau and the transcriptional repressor TDP-43, as well as the low-complexity motif in the nucleic-acid-binding protein FUS^{24,25,39,49,50,51,52,72,73}.

More recently, MicroED was used to shed light on HIV maturation, which involves the cleavage of the Gag capsid protein by protease⁷⁴. As Gag is cleaved, the capsid undergoes a conformational change from a sphere to a bullet shape, a hallmark indicator of a fully infectious HIV particle. Bevirimat is a known maturation inhibitor that binds to HIV Gag and prevents the cleavage by protease. However, the effectiveness of bevirimat is low, and efforts at further optimizing its drug-binding to HIV Gag were hindered by a lack of understanding of how bevirimat binds Gag and how it prevents protease cleavage. The structure of HIV-1 Gag was determined by X-ray crystallography, which produced a 3.2-Å structure⁷⁵; however, cocrystallization with bevirimat did not produce crystals, and incubation of preformed crystals with the drug led to severe deterioration of the crystals, thus preventing structure determination by X-ray crystallography. In contrast, nanocrystals of Gag diffracted in MicroED to 2.8 Å, and the addition of bevirimat to these preformed nanocrystals likewise yielded a 2.8-Å-resolution dataset. The MicroED structure of Gag with and without bevirimat indicates that a single bevirimat molecule bound at the sixfold axis of Gag acts as an allosteric inhibitor to prevent the cleavage of Gag by protease (Fig. 4b). Drug optimization can now begin to make bevirimat a better binder in the sixfold-symmetric binding pocket in order to increase the effectiveness of this inhibitor.

MicroED has also been applied to the study of inorganic nanomaterials to determine the structure of a ligand-capped gold nanoparticle⁴¹. Such gold clusters have important applications in nanotechnology, cancer therapy and targeted drug delivery. The structure of the gold cluster Au₁₄₆(p-MBA)₅₇ was determined by MicroED to 0.85-Å resolution, and the structure was confirmed by X-ray analysis, although at a lower resolution of 1.2 Å (XFELs delivered 1.5 Å only). The structure showed that the gold nanoparticle is organized as a twinned face-centered cubic cluster and revealed how the ligands interact with and stabilize the surface. Ordered rows of gold–hydrogen bonds were observed, shedding light on the mechanism by which this nanocage is stabilized (Fig. 4c). This work also demonstrated the importance of collecting samples under cryogenic conditions in a frozen-hydrated state when hydration is important, even for materials science samples. When the nanoparticle crystals were dehydrated before data collection, it was not possible to collect diffraction data from them. However, when the samples were prepared in the frozen-hydrated state by standard MicroED sample-preparation techniques, the crystals diffracted to very high resolution and the structure could be determined.

Opportunities for future development

Time-resolved structural studies

A significant advantage of using nanocrystals in crystallography is that time-resolved studies that involve the diffusion of molecules into the crystal are feasible and limited only by diffusion rates⁷⁶. Whereas the diffusion of small molecules into large crystals is inefficient (on the time scale of seconds when crystals have dimensions in the hundreds of micrometers), resulting in low occupancy and a lack of detail in density maps, diffusion into nanocrystals is extremely efficient (microsecond time scale), resulting in high occupancy and density maps with excellent details. Protein dynamics in electron crystallography have been studied for decades using 2D crystals^14,77,78,79, and similar protocols can be used for MicroED. The time resolution for these studies is limited to microsecond time scales by the rate of vitrification. While XFELs can look at reactions with femtosecond time resolution^80,81, many biological processes are much slower, taking place on microsecond to second time scales (for example, ligand binding and enzymatic reactions), thus providing an immense opportunity for time-resolved studies by MicroED. Synchrotrons can also be used to look at dynamics on these slower time scales, but the diffusion of ligands into large crystals is inefficient and slow, so the use of extremely small crystals and MicroED is a more attractive alternative. New sample-preparation tools and methods that allow fine control over the timing of sample mixing, application and blotting will greatly facilitate time-resolved MicroED studies, and these are ongoing areas of methods development. The ability of MicroED to determine structures from a small number of crystals would make these kinds of time-resolved studies much more efficient and feasible for systems where the quantity of available sample is limiting. Additionally, because electrons are sensitive to chemical bonding⁸², MicroED could one day be used to directly visualize the bonding interactions in a time-dependent experiment.

Conformational changes have been observed in MicroED experiments in at least two examples. For proteinase K, dynamics were observed in response to increasing exposure to the electron beam⁸³. At the start of the experiment, intact disulfide bridges were observed, but as the exposures increased, these bonds broke apart and the cysteine side chains moved away from one another (Fig. 8), which mirrors the observations seen in X-ray crystallography⁸⁴. In another example, also described above, the HIV Gag protein structure was determined with and without the maturation inhibitor bevirimat⁷⁴. This small molecule was added immediately before vitrification and could diffuse efficiently into preformed Gag nanocrystals before damaging the lattice, thus allowing the structures of both Gag alone and Gag–bevirimat to be determined to help with future drug-optimization efforts.

Fig. 8: Dynamics probed in response to radiation damage.

When less than 1 e^–/Ų (left) was used for structure determination of proteinase K (1.7 Å; PDB 6CL7), local radiation damage was minimal. When higher doses (right) were used (2.8 Å; PDB 6CLA), the damaging effects of the beam could be seen early on, with the breakage of disulfide bonds (red arrows) and lower attainable resolution. Density maps (2F_o–F_c) in gray are contoured at 1.5σ.

Realizing the damaging effects of the electron beam

Recently, a thorough analysis of radiation damage in cryo-EM was conducted using MicroED⁸³ (Fig. 8). Because sufficient data can be collected with exposures to ultralow electron doses, multiple complete high-resolution datasets can be collected from single crystals. Using very well-diffracting crystals of proteinase K, researchers showed that specific radiation damage was observed even with extremely low total exposures of ~1 e^–/Ų. A challenge for the future will be the creation of even more sensitive detectors and new electron sources to collect data faster, such that the additive effects of radiation damage become less significant while, at the same time, the necessary high-resolution data required for both cryo-EM imaging and diffraction techniques can be obtained.

Phasing methods in MicroED

Most MicroED structures have been determined via molecular replacement with homologous proteins used as search models^{8,23,24,25,48,71,83,85}. Direct methods were also successful in phasing MicroED data when the obtained resolution neared 1 Å (refs. ^39,49,72) with SHELX⁸⁶; however, this is applicable only in cases where the samples are exceptionally well diffracting. Therefore, a welcome development would be experimental phasing methods that can be used to determine novel structures where molecular replacement is not possible but the obtainable resolution is limited to ~2–3 Å. Imaging of the crystals and tomographic reconstructions are obvious steps forward, taking advantage of the unique properties of the TEM. Using images, a moderate-resolution molecular envelope of the sample within the crystal could be determined, which then could be used to phase the MicroED data. These efforts are currently under way in several laboratories^87,88. Heavy metal phasing is another avenue of active research. While this approach has been extremely successful in X-ray crystallography, no anomalous signal or absorption edge exists for compounds at the wavelengths of the electron microscope; therefore, only isomorphous replacement can be used for MicroED.

High-throughput small-molecule structure determination

As with biological molecules, electron diffraction can be extended to the study of organic molecules that form very small microcrystals^{40,82,89,90,91}. Recently, a study was presented that used MicroED to determine the atomic-resolution structures of 11 small molecules⁴⁰. These structures were determined directly from powders, with no additional crystallization optimization, and the process from sample preparation to structure solution was very quick. After the deposition of the samples onto the grid, data collection and processing were done in the same manner as described for biological crystals. The extension of MicroED to organic molecules offers a new analytical tool for studying the synthesis of new molecules. Future work that focuses on integrating the MicroED and organic synthesis pipelines promises to make this a routine method in chemistry labs.

Facilities, cameras and automation

As the use of cryo-EM methods such as MicroED becomes increasingly widespread, the need for additional centralized centers and facilities will increase. The equipment used for MicroED is essentially the same as for other cryo-EM modalities. For protein work, it is important to have an FEG, a sensitive and fast camera and, if possible, an energy filter. Camera technology is quickly progressing, and the use of direct electron detectors for MicroED is already starting. Cameras such as the Falcon3 should provide ultimate flexibility, as this camera could be used for all modalities in cryo-EM, including MicroED, whereas the other cameras appropriate for diffraction, such as the hybrid pixel detectors (for example, Timepix and EIGER^90,91,92), in their current configuration are not well suited for imaging and single-particle electron microscopy.

Automation is incredibly important and will be vital for MicroED, as it has greatly improved productivity for single-particle electron microscopy. Protocols and software already have been developed for MicroED automation, including an adaptation to SerialEM⁹³, but a complete suite with a friendly GUI is still lacking and is currently being developed by several laboratories. In the near future, users could ship samples to the user facilities and, after sample loading, could screen and collect MicroED data remotely from their home institutions, much like what is done at most X-ray synchrotron facilities. With the current implementation of SerialEM, more than 300 datasets can be collected overnight automatically⁹³. Because of the synergy among all modalities in cryo-EM user facilities, we expect that MicroED will have a broad impact on structural biology in the coming decades.