Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution, new validation tools, and implications for the future.

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) "Resolution Revolution" made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.

Structural Insights into Common and Host-Specific Receptor-Binding Mechanisms in Algal Picorna-like Viruses.

Marnaviridae viruses are abundant algal viruses that regulate the dynamics of algal blooms in aquatic environments. They employ a narrow host range because they merely lyse their algal host species. This host-specific lysis is thought to correspond to the unique receptor-binding mechanism of the Marnaviridae viruses. Here, we present the atomic structures of the full and empty capsids of Chaetoceros socialis forma radians RNA virus 1 built-in 3.0 Å and 3.1 Å cryo-electron microscopy maps. The empty capsid structure and the structural variability provide insights into its assembly and uncoating intermediates. In conjunction with the previously reported atomic model of the Chaetoceros tenuissimus RNA virus type II capsid, we have identified the common and diverse structural features of the VP1 surface between the Marnaviridae viruses. We have also tested the potential usage of AlphaFold2 for structural prediction of the VP1s and a subsequent structural phylogeny for classifying Marnaviridae viruses by their hosts. These findings will be crucial for inferring the host-specific receptor-binding mechanism in Marnaviridae viruses.

The diversity of protein-protein interaction interfaces within T=3 icosahedral viral capsids.

Some non-enveloped virus capsids assemble from multiple copies of a single type of coat-protein (CP). The comparative energetics of the diverse CP-CP interfaces present in such capsids likely govern virus assembly-disassembly mechanisms. The T = 3 icosahedral capsids comprise 180 CP copies arranged about two-, three-, five- and six-fold axes of (quasi-)rotation symmetry. Structurally diverse CPs can assemble into T = 3 capsids. Specifically, the Leviviridae CPs are structurally distinct from the Bromoviridae, Tombusviridae and Tymoviridae CPs which fold into the classic "jelly-roll" fold. However, capsids from across the four families are known to disassemble into dimers. To understand whether the overall symmetry of the capsid or the structural details of the CP determine virus assembly-disassembly mechanisms, we analyze the different CP-CP interfaces that occur in the four virus families. Previous work studied protein homodimer interfaces using interface size (relative to the monomer) and hydrophobicity. Here, we analyze all CP-CP interfaces using these two parameters and find that the dimerization interface (present between two CPs congruent through a two-fold axis of rotation) has a larger relative size in the Leviviridae than in the other viruses. The relative sizes of the other Leviviridae interfaces and all the jelly-roll interfaces are similar. However, the dimerization interfaces across families have slightly higher hydrophobicity, potentially making them stronger than other interfaces. Finally, although the CP-monomers of the jelly-roll viruses are structurally similar, differences in their dimerization interfaces leads to varied dimer flexibility. Overall, differences in CP-structures may induce different modes of swelling and assembly-disassembly in the T = 3 viruses.

MS2 and Qß bacteriophages reveal the contribution of surface hydrophobicity on the mobility of non-enveloped icosahedral viruses in SDS-based capillary zone electrophoresis.

SDS is commonly employed as BGE additive in CZE analysis of non-enveloped icosahedral viruses. But the way by which SDS interacts with the surface of such viruses remains to date poorly known, making complicate to understand their behavior during a run. In this article, two related bacteriophages, MS2 and Qß, are used as model to investigate the migration mechanism of non-enveloped icosahedral viruses in SDS-based CZE. Both phages are characterized by similar size and surface charge but significantly different surface hydrophobicity (Qß > MS2, where '>' means 'more hydrophobic than'). By comparing their electrophoretic mobility in the presence or not of SDS on both sides of the CMC, we show that surface hydrophobicity of phages is a key factor influencing their mobility and that SDS-virus association is driven by hydrophobic interactions at the surface of virions. The CZE analyses of heated MS2 particles, which over-express hydrophobic domains at their surface, confirm this finding. The correlations between the present results and others from the literature suggest that the proposed mechanism might not be exclusive to the bacteriophages examined here.

On the geometry of regular icosahedral capsids containing disymmetrons.

Large icosahedral virus capsids are composed of symmetrons, organized arrangements of capsomers. There are three types of symmetrons: disymmetrons, trisymmetrons, and pentasymmetrons, which have different shapes and are centered on the icosahedral 2-fold, 3-fold and 5-fold axes of symmetry, respectively. Sinkovits and Baker (2010) gave a classification of all possible ways of building an icosahedral structure solely from trisymmetrons and pentasymmetrons, which requires the triangulation number T to be odd. In the present paper we incorporate disymmetrons to obtain a geometric classification of large icosahedral viruses formed by regular penta-,tri-, and disymmetrons, giving all mathematically consistent and theoretically possible solutions. For every class of solutions, we further provide formulas for symmetron sizes and parity restrictions on h, k, and T numbers. We also present several methods in which invariants may be used to classify a given configuration.

Orbits of crystallographic embedding of non-crystallographic groups and applications to virology.

The architecture of infinite structures with non-crystallographic symmetries can be modelled via aperiodic tilings, but a systematic construction method for finite structures with non-crystallographic symmetry at different radial levels is still lacking. This paper presents a group theoretical method for the construction of finite nested point sets with non-crystallographic symmetry. Akin to the construction of quasicrystals, a non-crystallographic group G is embedded into the point group P of a higher-dimensional lattice and the chains of all G-containing subgroups are constructed. The orbits of lattice points under such subgroups are determined, and it is shown that their projection into a lower-dimensional G-invariant subspace consists of nested point sets with G-symmetry at each radial level. The number of different radial levels is bounded by the index of G in the subgroup of P. In the case of icosahedral symmetry, all subgroup chains are determined explicitly and it is illustrated that these point sets in projection provide blueprints that approximate the organization of simple viral capsids, encoding information on the structural organization of capsid proteins and the genomic material collectively, based on two case studies. Contrary to the affine extensions previously introduced, these orbits endow virus architecture with an underlying finite group structure, which lends itself better to the modelling of dynamic properties than its infinite-dimensional counterpart.

From an affine extended icosahedral group towards a toolkit for viral architecture.

The affine extensions (there are 55 different ones) of the icosahedral group developed by T. Keef and R. Twarock of the York Centre for Complex Systems Analysis of the University of York [see in particular Keef et al. (2013). Acta Cryst. A69, 140-150], and applied to the investigation of the architecture of a number of icosahedral viruses, are here considered in the framework of molecular crystallography. The basic ideas of such molecular description involve positions with rational indices which approximate backbone positions in viral polypeptide and RNA chains. The test case of the Pariacoto virus suggests that the best-fit algorithm used in the York group's approach should be adapted to a more specific toolkit suited for the investigation of the architecture of icosahedral viruses. Typical problems which could be solved by means of such a toolkit are exemplified and put in the perspective of viral properties.