cryo-EM Structure Determination Research Articles

Real-time cryo-EM structure determination

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Real-time cryo-EM structure determination for drug discovery

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Sub-3 Å Cryo-EM Structures of Necrosis Virus Particles via the Use of Multipurpose TEM with Electron Counting Camera.

During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes. Here, we investigated the capability of 200 kV multipurpose microscopes equipped with a direct electron camera in determining the structures of infectious particles. We used 30 nm particles of the grouper nerve necrosis virus as a test sample and obtained the cryo-EM structure with a resolution as high as ∼2.7 Å from a setting that used electron counting. For comparison, we tested a high-end cryo-EM (Talos Arctica) using a similar virus (Macrobrachium rosenbergii nodavirus) to obtain virtually the same resolution. Those results revealed that the resolution is ultimately limited by the depth of field. Our work updates the density maps of these viruses at the sub-3Å level to allow for building accurate atomic models from de novo to provide structural insights into the assembly of the capsids. Importantly, this study demonstrated that multipurpose TEMs are capable of the high-resolution cryo-EM structure determination of infectious particles and is thus germane to the research on pandemics.

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

Biochemical Society 2022 Award winners

Biochemical Society 2022 Award winners

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.

Structural determinants of cholesterol recognition in helical integral membrane proteins

Cholesterol is an integral component of mammalian membranes. It has been shown to modulate membrane fluidity and dynamics and alter integral membrane protein function. However, understanding the molecular mechanisms of how cholesterol impacts protein function is complicated by limited and conflicting structural data. Because of the nature of the crystallization and cryo-EM structure determination, it is difficult to distinguish between specific and biologically relevant interactions and a nonspecific association. The only widely recognized search algorithm for cholesterol-integral-membrane-protein interaction sites is sequence based, i.e., searching for the so-called "Cholesterol Recognition/interaction Amino acid Consensus" motif. Although these motifs are present in numerous integral membrane proteins, there is inconclusive evidence to support their necessity or sufficiency for cholesterol binding. Here, we leverage the increasing number of experimental cholesterol-integral-membrane-protein structures to systematically analyze putative interaction sites based on their spatial arrangement and evolutionary conservation. This analysis creates three-dimensional representations of general cholesterol interaction sites that form clusters across multiple integral membrane protein classes. We also classify cholesterol-integral-membrane-protein interaction sites as either likely-specific or nonspecific. Information gleaned from our characterization will eventually enable a structure-based approach to predict and design cholesterol-integral-membrane-protein interaction sites.

A Novel Chloride Conducting Conformation in Human Glutamate Transporters

Excitatory amino acid transporters (EAATs), which belong to the solute carrier 1A (SLC1A) family, couple glutamate transport to the co-transport of three sodium (Na+) ions and one proton (H+) and the counter-transport of one potassium (K+) ion. In addition to this coupled transport, binding of substrate and Na+ ions to EAATs activates a reversible and thermodynamically uncoupled chloride (Cl-) conductance. Mutations that alter this Cl- conductance have been linked to episodic ataxia type 6 (EA6). Structures of human EAATs and its homologues have highlighted that they share a similar trimeric structure and perform an elevator-like mechanism for the substrate transport, but a clear pathway for the uncoupled Cl- conductance has not been observed in the available structures. Therefore, the molecular determinants required for its activation remain unclear. Here, we present an extensive computational study, including microsecond-long molecular dynamics (MD) simulations and free-energy calculations, closely coupled to cryo-EM structure determination, electrophysiology, and mutagenesis to characterize a clear pathway for the uncoupled Cl- conductance in human and bacterial glutamate transporters. We have designed a modeling protocol which incorporates driven simulations combined with experimentally-derived restraints to capture the Cl- conducting conformation in human EAAT1. The free-energy calculations performed for the conduction of Cl- and Na+ ions through the captured conformation, highlight the presence of two hydrophobic gates which control the selective movement of Cl- through an aqueous-accessible permeation pathway. The computationally captured pathway was further validated by the cryo-EM structure of the Cl- conducting conformation in archaeal glutamate transporter (GlTph), mutagenesis, and functional studies. Overall, our findings provide insights into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

Cryo-EM structure determination captures new chemical modification of protein.

Cryo-EM structure determination captures new chemical modification of protein.

Exploiting prior knowledge about biological macromolecules in cryo-EM structure determination.

Three-dimensional reconstruction of the electron-scattering potential of biological macromolecules from electron cryo-microscopy (cryo-EM) projection images is an ill-posed problem. The most popular cryo-EM software solutions to date rely on a regularization approach that is based on the prior assumption that the scattering potential varies smoothly over three-dimensional space. Although this approach has been hugely successful in recent years, the amount of prior knowledge that it exploits compares unfavorably with the knowledge about biological structures that has been accumulated over decades of research in structural biology. Here, a regularization framework for cryo-EM structure determination is presented that exploits prior knowledge about biological structures through a convolutional neural network that is trained on known macromolecular structures. This neural network is inserted into the iterative cryo-EM structure-determination process through an approach that is inspired by regularization by denoising. It is shown that the new regularization approach yields better reconstructions than the current state of the art for simulated data, and options to extend this work for application to experimental cryo-EM data are discussed.

Helical Membrane Protein Crystallization in the New Era of Electron Cryo-Microscopy.

Helical assemblies of proteins, which consist of a two-dimensional lattice of identical subunits arranged with helical symmetry, are a common structural motif in nature. For membrane proteins, crystallization protocols can induce helical arrangements and take advantage of the symmetry found in these assemblies for the structural determination of target proteins. Modern advances in the field of electron cryo-microscopy (cryo-EM), in particular the advent of direct electron detectors, have opened the potential for structure determination of membrane proteins in such assemblies at high resolution. The nature of the symmetry in helical crystals of membrane proteins means that a single image potentially contains enough information for three-dimensional structural determination. With the current direct electron detectors, we have never been closer to making this a reality. Here, we present a protocol detailing the preparation of helical crystals, with an emphasis on further cryo-EM analysis and structural determination of the sarco(endo)plasmic reticulum Ca2+-ATPase in the presence of regulatory subunits such as phospholamban.

Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM.

Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air-water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

Expression and purification of the cardiac sodium channel NaV1.5 for cryo-EM structure determination.

Voltage-gated sodium channel NaV1.5 is responsible for initiating and propagating cardiac action potentials by selectively conducting Na+ into cardiomyocytes. Class-I antiarrhythmic drugs target NaV1.5 for treatment of arrhythmias. During the last few years, cryogenic electron microscopy (cryo-EM) has become a powerful technique to determine the structures of ion channels at atomic level. In order to reveal the structural features of NaV1.5 and the structural basis for its interaction with antiarrhythmic drugs by cryo-EM, NaV1.5 protein must be expressed at high levels and purified to homogeneity. In this chapter, we discuss the expression and purification of NaV1.5 in a mammalian expression system. We optimized the construct by deleting unstructured intracellular loops of rat NaV1.5 while retaining core functional regions. The resulting rNaV1.5C is fully functional and is blocked by Class-I antiarrhythmic drugs in a state-dependent manner. Protocols are presented for expressing and purifying sufficient sample of NaV1.5 for preparing cryo-EM grids. The resulting cryo-EM structure is briefly described.

Deep learning enables the atomic structure determination of the Fanconi Anemia core complex from cryoEM.

Cryo-electron microscopy of protein complexes often leads to moderate resolution maps (4-8 Å), with visible secondary-structure elements but poorly resolved loops, making model building challenging. In the absence of high-resolution structures of homologues, only coarse-grained structural features are typically inferred from these maps, and it is often impossible to assign specific regions of density to individual protein subunits. This paper describes a new method for overcoming these difficulties that integrates predicted residue distance distributions from a deep-learned convolutional neural network, computational protein folding using Rosetta, and automated EM-map-guided complex assembly. We apply this method to a 4.6 Å resolution cryoEM map of Fanconi Anemia core complex (FAcc), an E3 ubiquitin ligase required for DNA interstrand crosslink repair, which was previously challenging to interpret as it comprises 6557 residues, only 1897 of which are covered by homology models. In the published model built from this map, only 387 residues could be assigned to the specific subunits with confidence. By building and placing into density 42 deep-learning-guided models containing 4795 residues not included in the previously published structure, we are able to determine an almost-complete atomic model of FAcc, in which 5182 of the 6557 residues were placed. The resulting model is consistent with previously published biochemical data, and facilitates interpretation of disease-related mutational data. We anticipate that our approach will be broadly useful for cryoEM structure determination of large complexes containing many subunits for which there are no homologues of known structure.

Practical Considerations of Using the K3 Camera in CDS Mode for High-resolution and High-throughput Single Particle Cryo-EM

Abstract Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 A with a 1.346 A physical pixel). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 A physical pixel size) we achieved a resolution of 1.65 A within 9 hours of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 A resolution with 5 minutes of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.

Structural interpretation of cryo-EM image reconstructions

The productivity of single-particle cryo-EM as a structure determination method has rapidly increased as many novel biological structures are being elucidated. The ultimate result of the cryo-EM experiment is an atomic model that should faithfully represent the computed image reconstruction. Although the principal approach of atomic model building and refinement from maps resembles that of the X-ray crystallographic methods, there are important differences due to the unique properties resulting from the 3D image reconstructions. In this review, we discuss the practiced work-flow from the cryo-EM image reconstruction to the atomic model. We give an overview of (i) resolution determination methods in cryo-EM including local and directional resolution variation, (ii) cryo-EM map contrast optimization including complementary map types that can help in identifying ambiguous density features, (iii) atomic model building and (iv) refinement in various resolution regimes including (v) their validation and (vi) discuss differences between X-ray and cryo-EM maps. Based on the methods originally developed for X-ray crystallography, the path from 3D image reconstruction to atomic coordinates has become an integral and important part of the cryo-EM structure determination work-flow that routinely delivers atomic models.

Structural characterization of genomic RNA-coat protein contacts in single-stranded RNA viruses by high-resolution cryo-EM

Recent developments in cryo-electron microscopy (cryo-EM) hardware along with continuously evolving software tools have led to the discovery of many novel structures that it was not possible to solve until now, resulting in what is termed “the resolution revolution”. In structural virology, it has also led to a re-evaluation of known structures. Most virion structures solved by X-ray crystallography or cryo-EM are focused on the capsid protein (CP) as a result of the application of icosahedral symmetry averaging to “improve” the electron density maps. However, this has the consequence that the intrinsic asymmetry of important components of virions, such as the viral genome and structural proteins lacking such symmetry, are masked. Single-stranded (ss), positive-sense RNA viruses are major pathogens in all kingdoms of life. Asymmetric cryo-EM structure determination of a major model virus in this class, bacteriophage MS2, reveals the limitations of a symmetrized view. As well as the presence and interactions made by the unique Maturation Protein, it also reveals multiple gRNA-CP dimer contacts corresponding to our previous prediction that dispersed, sequence-degenerate RNA motifs (Packaging Signals, PSs) play important roles during the virion assembly. Here, we describe how relaxing symmetry during structure determination can image such gRNA-PS contacts in a range of ssRNA viruses including the picornavirus Bovine Enterovirus-1, the alphaviruses Sindbis and Semliki Forest Viruses, as well as the plant virus Turnip Crinkle Virus. The revelation of these functionally important gRNA-CP contacts changes our fundamental understanding of assembly in these pathogens and may have further translational importance.

High flavivirus structural plasticity demonstrated by a non-spherical morphological variant

Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion.

Synthetic antibodies against BRIL as universal fiducial marks for single\u2212particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

Cryo-EM Structure Determination and Model Fitting of the Proton-Gated Ligand-Gated Ion Channel glic at Multiple pH States

Cryo-EM Structure Determination and Model Fitting of the Proton-Gated Ligand-Gated Ion Channel glic at Multiple pH States