Single particle analysis integrated with microscopy: A high-throughput approach for reconstructing icosahedral particles

Autophagy is a highly regulated bulk degradation process that plays a key role in the maintenance of cellular homeostasis. During autophagy, a double membrane-bound compartment termed the autophagosome is formed through de novo nucleation and assembly of membrane sources to engulf unwanted cytoplasmic components and targets them to the lysosome or vacuole for degradation. Central to this process are the autophagy-related (ATG) proteins, which play a critical role in plant fitness, immunity, and environmental stress response. Over the past few years, cryo-electron microscopy (cryo-EM) and single-particle analysis has matured into a powerful and versatile technique for the structural determination of protein complexes at high resolution and has contributed greatly to our current understanding of the molecular mechanisms underlying autophagosome biogenesis. Here we describe the plant-specific ATG proteins and summarize recent structural and mechanistic studies on the protein machinery involved in autophagy initiation with an emphasis on those by single-particle analysis.

The structure of a T = 1 icosahedral particle (where T is the triangulation number), assembled from southern bean mosaic virus coat protein fragments that lacked the amino-terminal arm, was solved by means of model building procedures with the use of 6-angstrom resolution x-ray diffraction data. The icosahedral five-, three-, and twofold contacts were found to be similar, at this resolution, to the analogous contacts (icosahedral five-, quasi-three-, and quasi-twofolds) found in the parent T = 3 southern bean mosaic virus. However, the icosahedral fivefold contacts of the T = 3 structure are the most conserved in the T = 1 capsid. These results are consistent with a mechanism in which pentameric caps of dimers are the building blocks for the assembly of T = 1 and T = 3 icosahedral viruses.

Location of Auxilin Within a Clathrin Cage

Virus structures are megadalton nucleoprotein complexes with an exceptional variety of protein-protein and protein-nucleic-acid interactions. Three-dimensional crystal structures of over 70 virus capsids, from more than 20 families and 30 different genera of viruses, have been solved to near-atomic resolution. The enormous amount of information contained in these structures is difficult to access, even for scientists trained in structural biology. Virus Particle Explorer (VIPER) is a web-based catalogue of structural information that describes the icosahedral virus particles. In addition to high-resolution crystal structures, VIPER has expanded to include virus structures obtained by cryo-electron microscopy (EM) techniques. The VIPER database is a powerful resource for virologists, microbiologists, virus crystallographers and EM researchers. This review describes how to use VIPER, using several examples to show the power of this resource for research and educational purposes.

Rosellinia necatrix megabirnavirus 1-W779 (RnMBV1) is a non-enveloped icosahedral double-stranded (ds)RNA virus that infects the ascomycete fungus Rosellinia necatrix, a causative agent that induces a lethal plant disease white root rot. Herein, we have first resolved the atomic structure of the RnMBV1 capsid at 3.2 Å resolution using cryo-electron microscopy (cryo-EM) single-particle analysis. Compared with other non-enveloped icosahedral dsRNA viruses, the RnMBV1 capsid protein structure exhibits an extra-long C-terminal arm and a surface protrusion domain. In addition, the previously unrecognized crown proteins are identified in a symmetry-expanded cryo-EM model and are present over the 3-fold axes. These exclusive structural features of the RnMBV1 capsid could have been acquired for playing essential roles in transmission and/or particle assembly of the megabirnaviruses. Our findings, therefore, will reinforce the understanding of how the structural and molecular machineries of the megabirnaviruses influence the virulence of the disease-related ascomycete fungus.

Structure of the Connector of Bacteriophage T7 at 8 Å Resolution: Structural Homologies of a Basic Component of a DNA Translocating Machinery

A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM)). Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7-2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well.

Both single-particle analysis and electron tomography are special tools for the structural analysis of biological components by transmission electron microscopy. Single-particle analysis enables us to determine the structures of relatively large proteins and macromolecular complexes without crystallisation. Electron tomography is often used to visualise organelles and subcellular structures at molecular resolution. Recently, the scope of electron tomography has been widened due to the introduction of an automatic data collection system, cryo-electron microscopy, and improved sample preparation techniques. Application of electron tomography to single-particle analysis will be capable of analysing dynamics of large proteins. The techniques described in this review are expected to bridge the gap between the atomic resolution structures of protein molecules and cellular structures obtained by light microscopy.

Recent major advancements in cryo-electron microscopy (cryo-EM) have enabled high-resolution structural analysis, accompanied by developments in image processing software packages for single-particle analysis (SPA). SPA facilitates the 3D reconstruction of proteins and macromolecular complexes from numerous individual particles. In this study, we systematically evaluated the impact of symmetry parameters and particle quantity on the 3D reconstruction efficiency using the dihydrolipoyl acetyltransferase (E2) inner core of the pyruvate dehydrogenase complex (PDC). We specifically examined how inappropriate symmetry constraints can introduce structural artifacts and distortions, underscoring the necessity for accurate symmetry determination through rigorous validation methods such as directional Fourier shell correlation (FSC) and local-resolution mapping. Additionally, our analysis demonstrates that efficient reconstructions can be achieved with a moderate particle number, significantly reducing computational costs without compromising structural accuracy. We further contextualize these results by discussing recent developments in SPA workflows and hardware optimization, highlighting their roles in enhancing reconstruction accuracy and computational efficiency. Overall, our comprehensive benchmarking provides strategic insights that will facilitate the optimization of SPA experiments, particularly in resource-limited settings, and offers practical guidelines for accurately determining symmetry and particle quantity during cryo-EM data processing.

Rotaviruses, major causes of childhood gastroenteritis, are nonenveloped, icosahedral particles with double-strand RNA genomes. By the use of electron cryomicroscopy and single-particle reconstruction, we have visualized a rotavirus particle comprising the inner capsid coated with the trimeric outer-layer protein, VP7, at a resolution (4 A) comparable with that of X-ray crystallography. We have traced the VP7 polypeptide chain, including parts not seen in its X-ray crystal structure. The 3 well-ordered, 30-residue, N-terminal "arms" of each VP7 trimer grip the underlying trimer of VP6, an inner-capsid protein. Structural differences between free and particle-bound VP7 and between free and VP7-coated inner capsids may regulate mRNA transcription and release. The Ca(2+)-stabilized VP7 intratrimer contact region, which presents important neutralizing epitopes, is unaltered upon capsid binding.

In cryo-electron microscopy (cryo-EM), the Wiener filter is the optimal operation – in the least-squares sense – of merging a set of aligned low signal-to-noise ratio (SNR) micrographs to obtain a class average image with higher SNR. However, the condition for the optimal behavior of the Wiener filter is that the signal of interest shows stationary characteristic thoroughly, which cannot always be satisfied. In this paper, we propose substituting the conventional Wiener filter, which encompasses the whole image for denoising, with its local adaptive implementation, which denoises the signal locally. We compare our proposed local adaptive Wiener filter (LA-Wiener filter) with the conventional class averaging method using a simulated dataset and an experimental cryo-EM dataset. The visual and numerical analyses of the results indicate that LA-Wiener filter is superior to the conventional approach in single particle reconstruction (SPR) applications.

Efforts in structural biology have targeted the systematic determination of all protein structures through experimental determination or modeling. In recent years, 3-D electron cryomicroscopy (cryoEM) has assumed an increasingly important role in determining the structures of these large macromolecular assemblies to intermediate resolutions (6–10 Å). While these structures provide a snapshot of the assembly and its components in well-defined functional states, the resolution limits the ability to build accurate structural models. In contrast, sequence-based modeling techniques are capable of producing relatively robust structural models for isolated proteins or domains. In this work, we developed and applied a hybrid modeling approach, utilizing cryoEM density and ab initio modeling to produce a structural model for the core domain of a herpesvirus structural protein, VP26. Specifically, this method, first tested on simulated data, utilizes the cryoEM density map as a geometrical constraint in identifying the most native-like models from a gallery of models generated by ab initio modeling. The resulting model for the core domain of VP26, based on the 8.5-Å resolution herpes simplex virus type 1 (HSV-1) capsid cryoEM structure and mutational data, exhibited a novel fold. Additionally, the core domain of VP26 appeared to have a complementary interface to the known upper-domain structure of VP5, its cognate binding partner. While this new model provides for a better understanding of the assembly and interactions of VP26 in HSV-1, the approach itself may have broader applications in modeling the components of large macromolecular assemblies.

DNA replication is a key cellular process and is the basis for cell division. In order to understand replication initiation in mammalian cells, viral systems such as bovine papillomavirus (BPV) are used as simplified models of the process. BPV uses a single protein E1 to perform both DNA binding and unwinding functions essential for the initiation of replication (Figure A). Biochemical assays for a cell‐free replication initiation system has been established for BPV. To have a clearer understanding of a biological process at a molecular level, it is essential to determine the three‐dimensional (3D) arrangement and dynamics of molecules and how macromolecular machines are assembled. Electron microscopy (EM), single particle analysis and image processing have been used in this work to reveal the structures of the BPV E1 helicase domain (E1HD), full‐length E1 (E1FL), a single‐labelled E1FL‐replication fork DNA complex with monovalent tetrameric streptavidin (MTS) on the dsDNA of the fork and a double‐labelled E1FLwith MTS on the dsDNA and FAB on the ssDNA of the fork. The structure of BPV E1FL in complex with DNA was initially determined by EM both by applying 6‐fold symmetry and without applying any symmetry. EM demonstrated that the E1FL helicase forms a hexamer that has a diameter of 130Å and a height of 100Å, consistent with an overall mass of ~410 kDa. The oligomer has a central channel inside the molecule, with a variable diameter. E1FL is a single polypeptide chain, and the OBD domain can be fitted into the structure in two different ways. In one case the DNA binding site would be located on the inner surface and in the other case it would be on the outer surface of the complex. To determine the orientation of the OBD domain, a 12‐residue epitope sequence GGYPYDVPDYAG was inserted into the OBD domain after the residue 226. EM has demonstrated that the antibody was bound to the E1FL. That proves that the binding site for AB is located on the outer surface of E1FL (Figure B). The points of the entrance of dsDNA and exit of 5′ ssDNA were also determined in the complex of E1FL and DNA. Labelling with streptavidin was performed to reveal the position of the dsDNA, whereas ssDNA was labelled with DIG‐FAB to reveal the 5′ ssDNA position in an asymmetrical structure of the BPV E1 hexamer bound to a replication fork DNA substrate. Comparison of the 3D structures showed that dsDNA enters the molecule between the N‐terminus and ori DNA‐binding domain (OBD), and the 5′ ssDNA exits the molecule between the collar domain and OBD. The angle between the point of dsDNA entrance and 5′ ssDNA exit was ~170°. Prior to our research, the most accepted model of “steric exclusion” for dsDNA unwinding suggested that the active 3′ ssDNA strand is pulled through the helicase motor and dsDNA is wedged apart outside the protein assembly (Figure C). Our structural observations indicated that strand separation is taking place inside E1 in a chamber above the helicase domain and that the 5′ passive ssDNA strand leaves the assembly through a channel located on the opposite side to dsDNA entry. Therefore, our data suggest an alternative model for DNA unwinding by this general class of replication enzymes.

In the recent years, cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) has greatly benefited from a multitude of technological advances cumulating in breaking the atomic-resolution barrier for biological macromolecules. Since then, structural insights by cryo-EM have led to driving rational design of new vaccines and therapeutics due to high throughput and high-resolution screening and data collection technologies. Additionally, cryo-electron tomography (cryo-ET) has become an indispensable tool for structural cell biology. In this presentation we elaborate on the latest Thermo Fisher Scientific innovations for 300kV cryo electron microscopy for SPA and cryo-ET to advance more workflow efficiency and performance. Our ongoing mission is to further improve the connectivity between the cryo-FIB and cryo-TEM for an even more streamlined tomography workflow that is safer, faster and cleaner. Data acquisition and processing can be a significant cryo-ET bottleneck. Our integrated solution, combining Thermo Scientific Tomography 5 and Tomo Live, automates pre-acquisition tasks, enhances productivity with predictive tracking and enables rapid quality assessment. Robust and optimized motion correction helps produce crisp tomograms and image quality needed for sub-tomogram averaging. Results are displayed with new quality metrics on a web platform, making the workflow user-friendly and facilitating structural biology advancements.

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.