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.

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity.

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

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.

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.

SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.

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.

Ligand recognition and G-protein coupling selectivity of cholecystokinin A receptor.

Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Gi and Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Gi and Gq heterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the 'wavy hook' of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.

Cryo-EM structure of the Rhodospirillum rubrum RC-LH1 complex at 2.5†Å.

The reaction centre light-harvesting 1 (RC-LH1) complex is the core functional component of bacterial photosynthesis. We determined the cryo-electron microscopy (cryo-EM) structure of the RC-LH1 complex from Rhodospirillum rubrum at 2.5 Šresolution, which reveals a unique monomeric bacteriochlorophyll with a phospholipid ligand in the gap between the RC and LH1 complexes. The LH1 complex comprises a circular array of 16 αß-polypeptide subunits that completely surrounds the RC, with a preferential binding site for a quinone, designated QP, on the inner face of the encircling LH1 complex. Quinols, initially generated at the RC QB site, are proposed to transiently occupy the QP site prior to traversing the LH1 barrier and diffusing to the cytochrome bc1 complex. Thus, the QP site, which is analogous to other such sites in recent cryo-EM structures of RC-LH1 complexes, likely reflects a general mechanism for exporting quinols from the RC-LH1 complex.

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 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.

Improving SARS-CoV-2 structures: Peer review by early coordinate release.

This work builds upon the record-breaking speed and generous immediate release of new experimental three-dimensional structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and complexes, which are crucial to downstream vaccine and drug development. We have surveyed those structures to catch the occasional errors that could be significant for those important uses and for which we were able to provide demonstrably higher-accuracy corrections. This process relied on new validation and correction methods such as CaBLAM and ISOLDE, which are not yet in routine use. We found such important and correctable problems in seven early SARS-CoV-2 structures. Two of the structures were soon superseded by new higher-resolution data, confirming our proposed changes. For the other five, we emailed the depositors a documented and illustrated report and encouraged them to make the model corrections themselves and use the new option at the worldwide Protein Data Bank for depositors to re-version their coordinates without changing the Protein Data Bank code. This quickly and easily makes the better-accuracy coordinates available to anyone who examines or downloads their structure, even before formal publication. The changes have involved sequence misalignments, incorrect RNA conformations near a bound inhibitor, incorrect metal ligands, and cis-trans or peptide flips that prevent good contact at interaction sites. These improvements have propagated into nearly all related structures done afterward. This process constitutes a new form of highly rigorous peer review, which is actually faster and more strict than standard publication review because it has access to coordinates and maps; journal peer review would also be strengthened by such access.

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.

Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55.

BTB-Kelch proteins are substrate-specific adaptors for cullin-3 (Cul3) RING-box-based E3 ubiquitin ligases, mediating protein ubiquitylation for subsequent proteasomal degradation. Vaccinia virus encodes three BTB-Kelch proteins: A55, C2, and F3. Viruses lacking A55 or C2 have altered cytopathic effects in cultured cells and altered pathology in vivo Previous studies have shown that the ectromelia virus orthologue of A55 interacts with Cul3 in cells. We report that the N-terminal BTB-BACK (BB) domain of A55 binds directly to the Cul3 N-terminal domain (Cul3-NTD), forming a 2:2 complex in solution. We solved the structure of an A55BB/Cul3-NTD complex from anisotropic crystals diffracting to 2.3/3.7 Å resolution in the best/worst direction, revealing that the overall interaction and binding interface closely resemble the structures of cellular BTB/Cul3-NTD complexes, despite low sequence identity between A55 and cellular BTB domains. Surprisingly, despite this structural similarity, the affinity of Cul3-NTD for A55BB was stronger than for cellular BTB proteins. Glutamate substitution of the A55 residue Ile-48, adjacent to the canonical φX(D/E) Cul3-binding motif, reduced affinity of A55BB for Cul3-NTD by at least 2 orders of magnitude. Moreover, Ile-48 and the φX(D/E) motif are conserved in A55 orthologues from other poxviruses, but not in the vaccinia virus proteins C2 or F3. The high-affinity interaction between A55BB and Cul3-NTD suggests that, in addition to directing the Cul3-RING E3 ligase complex to degrade cellular/viral target proteins that are normally unaffected, A55 may also sequester Cul3 from cellular adaptor proteins, thereby protecting substrates of these cellular adaptors from ubiquitylation and degradation.

Multiple functional self-association interfaces in plant TIR domains.

The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

Evaluation of template-based modeling in CASP13.

Performance in the template-based modeling (TBM) category of CASP13 is assessed here, using a variety of metrics. Performance of the predictor groups that participated is ranked using the primary ranking score that was developed by the assessors for CASP12. This reveals that the best results are obtained by groups that include contact predictions or inter-residue distance predictions derived from deep multiple sequence alignments. In cases where there is a good homolog in the wwPDB (TBM-easy category), the best results are obtained by modifying a template. However, for cases with poorer homologs (TBM-hard), very good results can be obtained without using an explicit template, by deep learning algorithms trained on the wwPDB. Alternative metrics are introduced, to allow testing of aspects of structural models that are not addressed by traditional CASP metrics. These include comparisons to the main-chain and side-chain torsion angles of the target, and the utility of models for solving crystal structures by the molecular replacement method. The alternative metrics are poorly correlated with the traditional metrics, and it is proposed that modeling has reached a sufficient level of maturity that the best models should be expected to satisfy this wider range of criteria.

Evaluation of model refinement in CASP13.

Performance in the model refinement category of the 13th round of Critical Assessment of Structure Prediction (CASP13) is assessed, showing that some groups consistently improve most starting models whereas the majority of participants continue to degrade the starting model on average. Using the ranking formula developed for CASP12, it is shown that only 7 of 32 groups perform better than a "naïve predictor" who just submits the starting model. Common features in their approaches include a dependence on physics-based force fields to judge alternative conformations and the use of molecular dynamics to relax models to local minima, usually with some restraints to prevent excessively large movements. In addition to the traditional CASP metrics that focus largely on the quality of the overall fold, alternative metrics are evaluated, including comparisons of the main-chain and side-chain torsion angles, and the utility of the models for solving crystal structures by the molecular replacement method. It is proposed that the introduction of these metrics, as well as consideration of the accuracy of coordinate error estimates, would improve the discrimination between good and very good models.

A structural analysis of DNA binding by hSSB1 (NABP2/OBFC2B) in solution.

Single-stranded DNA binding proteins (SSBs) play an important role in DNA processing events such as replication, recombination and repair. Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) contains a single oligosaccharide/oligonucleotide binding (OB) domain followed by a charged C-terminus and is structurally homologous to the SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus Recent work has revealed that hSSB1 is critical to homologous recombination and numerous other important biological processes such as the regulation of telomeres, the maintenance of DNA replication forks and oxidative damage repair. Since the ability of hSSB1 to directly interact with single-stranded DNA (ssDNA) is paramount for all of these processes, understanding the molecular details of ssDNA recognition is essential. In this study, we have used solution-state nuclear magnetic resonance in combination with biophysical and functional experiments to structurally analyse ssDNA binding by hSSB1. We reveal that ssDNA recognition in solution is modulated by base-stacking of four key aromatic residues within the OB domain. This DNA binding mode differs significantly from the recently determined crystal structure of the SOSS1 complex containing hSSB1 and ssDNA. Our findings elucidate the detailed molecular mechanism in solution of ssDNA binding by hSSB1, a major player in the maintenance of genomic stability.