EMinsight: a tool to capture cryoEM microscope configuration and experimental outcomes for analysis and deposition.

The widespread adoption of cryoEM technologies for structural biology has pushed the discipline to new frontiers. A significant worldwide effort has refined the single-particle analysis (SPA) workflow into a reasonably standardized procedure. Significant investments of development time have been made, particularly in sample preparation, microscope data-collection efficiency, pipeline analyses and data archiving. The widespread adoption of specific commercial microscopes, software for controlling them and best practices developed at facilities worldwide has also begun to establish a degree of standardization to data structures coming from the SPA workflow. There is opportunity to capitalize on this moment in the maturation of the field, to capture metadata from SPA experiments and correlate the metadata with experimental outcomes, which is presented here in a set of programs called EMinsight. This tool aims to prototype the framework and types of analyses that could lead to new insights into optimal microscope configurations as well as to define methods for metadata capture to assist with the archiving of cryoEM SPA data. It is also envisaged that this tool will be useful to microscope operators and facilities looking to rapidly generate reports on SPA data-collection and screening sessions.

A service-based approach to cryoEM facility processing pipelines at eBIC.

Electron cryo-microscopy image-processing workflows are typically composed of elements that may, broadly speaking, be categorized as high-throughput workloads which transition to high-performance workloads as preprocessed data are aggregated. The high-throughput elements are of particular importance in the context of live processing, where an optimal response is highly coupled to the temporal profile of the data collection. In other words, each movie should be processed as quickly as possible at the earliest opportunity. The high level of disconnected parallelization in the high-throughput problem directly allows a completely scalable solution across a distributed computer system, with the only technical obstacle being an efficient and reliable implementation. The cloud computing frameworks primarily developed for the deployment of high-availability web applications provide an environment with a number of appealing features for such high-throughput processing tasks. Here, an implementation of an early-stage processing pipeline for electron cryotomography experiments using a service-based architecture deployed on a Kubernetes cluster is discussed in order to demonstrate the benefits of this approach and how it may be extended to scenarios of considerably increased complexity.

Computational models of amorphous ice for accurate simulation of cryo-EM images of biological samples.

Simulations of cryo-electron microscopy (cryo-EM) images of biological samples can be used to produce test datasets to support the development of instrumentation, methods, and software, as well as to assess data acquisition and analysis strategies. To be useful, these simulations need to be based on physically realistic models which include large volumes of amorphous ice. The gold standard model for EM image simulation is a physical atom-based ice model produced using molecular dynamics simulations. Although practical for small sample volumes; for simulation of cryo-EM data from large sample volumes, this can be too computationally expensive. We have evaluated a Gaussian Random Field (GRF) ice model which is shown to be more computationally efficient for large sample volumes. The simulated EM images are compared with the gold standard atom-based ice model approach and shown to be directly comparable. Comparison with experimentally acquired data shows the Gaussian random field ice model produces realistic simulations. The software required has been implemented in the Parakeet software package and the underlying atomic models are available online for use by the wider community.

A review of the approaches used to solve sub-100 kDa membrane proteins by cryo-electron microscopy.

Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structurally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity as well as conformational and compositional instability. Here we have reviewed the sample preparation approaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM (those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.

Cryo-plasma FIB/SEM volume imaging of biological specimens.

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM. We evaluated the choice of plasma ion source and imaging regimes to produce high-quality SEM images of a range of different biological samples. Using an automated workflow we produced three-dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20-50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.

Application of super-resolution and correlative double sampling in cryo-electron microscopy.

Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that collecting cryo-ET STA data using the same conditions as SPA, with both correlated double sampling (CDS) and the super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in the super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in the super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising the data size on disk and the area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).

Approaches to Using the Chameleon: Robust, Automated, Fast-Plunge cryoEM Specimen Preparation.

The specimen preparation process is a key determinant in the success of any cryo electron microscopy (cryoEM) structural study and until recently had remained largely unchanged from the initial designs of Jacques Dubochet and others in the 1980s. The process has transformed structural biology, but it is largely manual and can require extensive optimisation for each protein sample. The chameleon instrument with its self-wicking grids and fast-plunge freezing represents a shift towards a robust, automated, and highly controllable future for specimen preparation. However, these new technologies require new workflows and an understanding of their limitations and strengths. As early adopters of the chameleon technology, we report on our experiences and lessons learned through case studies. We use these to make recommendations for the benefit of future users of the chameleon system and the field of cryoEM specimen preparation generally.

Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes.

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.

High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.

Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.

The antibody response to SARS-CoV-2 Beta underscores the antigenic distance to other variants.

Alpha-B.1.1.7, Beta-B.1.351, Gamma-P.1, and Delta-B.1.617.2 variants of SARS-CoV-2 express multiple mutations in the spike protein (S). These may alter the antigenic structure of S, causing escape from natural or vaccine-induced immunity. Beta is particularly difficult to neutralize using serum induced by early pandemic SARS-CoV-2 strains and is most antigenically separated from Delta. To understand this, we generated 674 mAbs from Beta-infected individuals and performed a detailed structure-function analysis of the 27 most potent mAbs: one binding the spike N-terminal domain (NTD), the rest the receptor-binding domain (RBD). Two of these RBD-binding mAbs recognize a neutralizing epitope conserved between SARS-CoV-1 and -2, while 18 target mutated residues in Beta: K417N, E484K, and N501Y. There is a major response to N501Y, including a public IgVH4-39 sequence, with E484K and K417N also targeted. Recognition of these key residues underscores why serum from Beta cases poorly neutralizes early pandemic and Delta viruses.

Parakeet: a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography.

In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a 'digital twin' software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.

A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19.

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

CryoEM structure of the super-constricted two-start dynamin 1 filament.

Dynamin belongs to the large GTPase superfamily, and mediates the fission of vesicles during endocytosis. Dynamin molecules are recruited to the neck of budding vesicles to assemble into a helical collar and to constrict the underlying membrane. Two helical forms were observed: the one-start helix in the constricted state and the two-start helix in the super-constricted state. Here we report the cryoEM structure of a super-constricted two-start dynamin 1 filament at 3.74 Å resolution. The two strands are joined by the conserved GTPase dimeric interface. In comparison with the one-start structure, a rotation around Hinge 1 is observed, essential for communicating the chemical power of the GTPase domain and the mechanical force of the Stalk and PH domain onto the underlying membrane. The Stalk interfaces are well conserved and serve as fulcrums for adapting to changing curvatures. Relative to one-start, small rotations per interface accumulate to bring a drastic change in the helical pitch. Elasticity theory rationalizes the diversity of dynamin helical symmetries and suggests corresponding functional significance.

Single Particle Cryo-Electron Microscopy: From Sample to Structure.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

The antigenic anatomy of SARS-CoV-2 receptor binding domain.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

Author Correction: Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Assembly intermediates of orthoreovirus captured in the cell.

Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected 'single shelled' intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is 'collapsed' compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.