Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

AlphaFold predictions are valuable hypotheses and accelerate but do not replace experimental structure determination.

Artificial intelligence-based protein structure prediction methods such as AlphaFold have revolutionized structural biology. The accuracies of these predictions vary, however, and they do not take into account ligands, covalent modifications or other environmental factors. Here, we evaluate how well AlphaFold predictions can be expected to describe the structure of a protein by comparing predictions directly with experimental crystallographic maps. In many cases, AlphaFold predictions matched experimental maps remarkably closely. In other cases, even very high-confidence predictions differed from experimental maps on a global scale through distortion and domain orientation, and on a local scale in backbone and side-chain conformation. We suggest considering AlphaFold predictions as exceptionally useful hypotheses. We further suggest that it is important to consider the confidence in prediction when interpreting AlphaFold predictions and to carry out experimental structure determination to verify structural details, particularly those that involve interactions not included in the prediction.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography.

Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.

The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome.

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two sub-assemblies: Retriever composed of VPS35L, VPS26C, and VPS29; and the CCC complex which contains twelve subunits: COMMD1-COMMD10 and the coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy, and in silico predictions, we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilized by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a 16th subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Accelerating crystal structure determination with iterative AlphaFold prediction.

Experimental structure determination can be accelerated with artificial intelligence (AI)-based structure-prediction methods such as AlphaFold. Here, an automatic procedure requiring only sequence information and crystallographic data is presented that uses AlphaFold predictions to produce an electron-density map and a structural model. Iterating through cycles of structure prediction is a key element of this procedure: a predicted model rebuilt in one cycle is used as a template for prediction in the next cycle. This procedure was applied to X-ray data for 215 structures released by the Protein Data Bank in a recent six-month period. In 87% of cases our procedure yielded a model with at least 50% of Cα atoms matching those in the deposited models within 2 Å. Predictions from the iterative template-guided prediction procedure were more accurate than those obtained without templates. It is concluded that AlphaFold predictions obtained based on sequence information alone are usually accurate enough to solve the crystallographic phase problem with molecular replacement, and a general strategy for macromolecular structure determination that includes AI-based prediction both as a starting point and as a method of model optimization is suggested.

Putting AlphaFold models to work with phenix.process_predicted_model and ISOLDE.

AlphaFold has recently become an important tool in providing models for experimental structure determination by X-ray crystallography and cryo-EM. Large parts of the predicted models typically approach the accuracy of experimentally determined structures, although there are frequently local errors and errors in the relative orientations of domains. Importantly, residues in the model of a protein predicted by AlphaFold are tagged with a predicted local distance difference test score, informing users about which regions of the structure are predicted with less confidence. AlphaFold also produces a predicted aligned error matrix indicating its confidence in the relative positions of each pair of residues in the predicted model. The phenix.process_predicted_model tool downweights or removes low-confidence residues and can break a model into confidently predicted domains in preparation for molecular replacement or cryo-EM docking. These confidence metrics are further used in ISOLDE to weight torsion and atom-atom distance restraints, allowing the complete AlphaFold model to be interactively rearranged to match the docked fragments and reducing the need for the rebuilding of connecting regions.

A structural biology community assessment of AlphaFold2 applications.

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods for protein structure predictions have reached the accuracy of experimentally determined models. Although this has been independently verified, the implementation of these methods across structural-biology applications remains to be tested. Here, we evaluate the use of AlphaFold2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modeling of interactions; and modeling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modeled when compared with homology modeling, identifying structural features rarely seen in the Protein Data Bank. AF2-based predictions of protein disorder and complexes surpass dedicated tools, and AF2 models can be used across diverse applications equally well compared with experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life-science research.

Improved AlphaFold modeling with implicit experimental information.

Machine-learning prediction algorithms such as AlphaFold and RoseTTAFold can create remarkably accurate protein models, but these models usually have some regions that are predicted with low confidence or poor accuracy. We hypothesized that by implicitly including new experimental information such as a density map, a greater portion of a model could be predicted accurately, and that this might synergistically improve parts of the model that were not fully addressed by either machine learning or experiment alone. An iterative procedure was developed in which AlphaFold models are automatically rebuilt on the basis of experimental density maps and the rebuilt models are used as templates in new AlphaFold predictions. We show that including experimental information improves prediction beyond the improvement obtained with simple rebuilding guided by the experimental data. This procedure for AlphaFold modeling with density has been incorporated into an automated procedure for interpretation of crystallographic and electron cryo-microscopy maps.

Cryo-EM structures of light-harvesting 2 complexes from Rhodopseudomonas palustris reveal the molecular origin of absorption tuning.

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and ß-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris, each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αß-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αß-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor.

Monomers of the insulin receptor and type 1 insulin-like growth factor receptor (IGF-1R) can combine stochastically to form heterodimeric hybrid receptors. These hybrid receptors display ligand binding and signaling properties that differ from those of the homodimeric receptors. Here, we describe the cryoelectron microscopy structure of such a hybrid receptor in complex with insulin-like growth factor I (IGF-I). The structure (ca. 3.7 Å resolution) displays a single IGF-I ligand, bound in a similar fashion to that seen for IGFs in complex with IGF-1R. The IGF-I ligand engages the first leucine-rich-repeat domain and cysteine-rich region of the IGF-1R monomer (rather than those of the insulin receptor monomer), consistent with the determinants for IGF binding residing in the IGF-1R cysteine-rich region. The structure broadens our understanding of this receptor family and assists in delineating the key structural motifs involved in binding their respective ligands.

2.4-Å structure of the double-ring Gemmatimonas phototrophica photosystem.

Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.

Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.

Cryo-EM structure of the dimeric Rhodobacter sphaeroides RC-LH1 core complex at 2.9†Å: the structural basis for dimerisation.

The dimeric reaction centre light-harvesting 1 (RC-LH1) core complex of Rhodobacter sphaeroides converts absorbed light energy to a charge separation, and then it reduces a quinone electron and proton acceptor to a quinol. The angle between the two monomers imposes a bent configuration on the dimer complex, which exerts a major influence on the curvature of the membrane vesicles, known as chromatophores, where the light-driven photosynthetic reactions take place. To investigate the dimerisation interface between two RC-LH1 monomers, we determined the cryogenic electron microscopy structure of the dimeric complex at 2.9 Šresolution. The structure shows that each monomer consists of a central RC partly enclosed by a 14-subunit LH1 ring held in an open state by PufX and protein-Y polypeptides, thus enabling quinones to enter and leave the complex. Two monomers are brought together through N-terminal interactions between PufX polypeptides on the cytoplasmic side of the complex, augmented by two novel transmembrane polypeptides, designated protein-Z, that bind to the outer faces of the two central LH1 ß polypeptides. The precise fit at the dimer interface, enabled by PufX and protein-Z, by C-terminal interactions between opposing LH1 αß subunits, and by a series of interactions with a bound sulfoquinovosyl diacylglycerol lipid, bring together each monomer creating an S-shaped array of 28 bacteriochlorophylls. The seamless join between the two sets of LH1 bacteriochlorophylls provides a path for excitation energy absorbed by one half of the complex to migrate across the dimer interface to the other half.

Cryo-EM Structure of the Rhodobacter sphaeroides Light-Harvesting 2 Complex at 2.1 Å.

Light-harvesting 2 (LH2) antenna complexes augment the collection of solar energy in many phototrophic bacteria. Despite its frequent role as a model for such complexes, there has been no three-dimensional (3D) structure available for the LH2 from the purple phototroph Rhodobacter sphaeroides. We used cryo-electron microscopy (cryo-EM) to determine the 2.1 Å resolution structure of this LH2 antenna, which is a cylindrical assembly of nine αß heterodimer subunits, each of which binds three bacteriochlorophyll a (BChl) molecules and one carotenoid. The high resolution of this structure reveals all of the interpigment and pigment-protein interactions that promote the assembly and energy-transfer properties of this complex. Near the cytoplasmic face of the complex there is a ring of nine BChls, which absorb maximally at 800 nm and are designated as B800; each B800 is coordinated by the N-terminal carboxymethionine of LH2-α, part of a network of interactions with nearby residues on both LH2-α and LH2-ß and with the carotenoid. Nine carotenoids, which are spheroidene in the strain we analyzed, snake through the complex, traversing the membrane and interacting with a ring of 18 BChls situated toward the periplasmic side of the complex. Hydrogen bonds with C-terminal aromatic residues modify the absorption of these pigments, which are red-shifted to 850 nm. Overlaps between the macrocycles of the B850 BChls ensure rapid transfer of excitation energy around this ring of pigments, which act as the donors of energy to neighboring LH2 and reaction center light-harvesting 1 (RC-LH1) complexes.

Cryo-EM structure of the monomeric Rhodobacter sphaeroides RC-LH1 core complex at 2.5†Å.

Reaction centre light-harvesting 1 (RC-LH1) complexes are the essential components of bacterial photosynthesis. The membrane-intrinsic LH1 complex absorbs light and the energy migrates to an enclosed RC where a succession of electron and proton transfers conserves the energy as a quinol, which is exported to the cytochrome bc1 complex. In some RC-LH1 variants quinols can diffuse through small pores in a fully circular, 16-subunit LH1 ring, while in others missing LH1 subunits create a gap for quinol export. We used cryogenic electron microscopy to obtain a 2.5 Šresolution structure of one such RC-LH1, a monomeric complex from Rhodobacter sphaeroides. The structure shows that the RC is partly enclosed by a 14-subunit LH1 ring in which each αß heterodimer binds two bacteriochlorophylls and, unusually for currently reported complexes, two carotenoids rather than one. Although the extra carotenoids confer an advantage in terms of photoprotection and light harvesting, they could impede passage of quinones through small, transient pores in the LH1 ring, necessitating a mechanism to create a dedicated quinone channel. The structure shows that two transmembrane proteins play a part in stabilising an open ring structure; one of these components, the PufX polypeptide, is augmented by a hitherto undescribed protein subunit we designate as protein-Y, which lies against the transmembrane regions of the thirteenth and fourteenth LH1α polypeptides. Protein-Y prevents LH1 subunits 11-14 adjacent to the RC QB site from bending inwards towards the RC and, with PufX preventing complete encirclement of the RC, this pair of polypeptides ensures unhindered quinone diffusion.