The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein.

Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.

EIGER2 hybrid-photon-counting X-ray detectors for advanced synchrotron diffraction experiments.

The ability to utilize a hybrid-photon-counting detector to its full potential can significantly influence data quality, data collection speed, as well as development of elaborate data acquisition schemes. This paper facilitates the optimal use of EIGER2 detectors by providing theory and practical advice on (i) the relation between detector design, technical specifications and operating modes, (ii) the use of corrections and calibrations, and (iii) new acquisition features: a double-gating mode, 8-bit readout mode for increasing temporal resolution, and lines region-of-interest readout mode for frame rates up to 98 kHz. Examples of the implementation and application of EIGER2 at several synchrotron sources (ESRF, PETRA III/DESY, ELETTRA, AS/ANSTO) are presented: high accuracy of high-throughput data in serial crystallography using hard X-rays; suppressing higher harmonics of undulator radiation, improving peak shapes, increasing data collection speed in powder X-ray diffraction; faster ptychography scans; and cleaner and faster pump-and-probe experiments.

Crystal structure of the RNA-dependent RNA polymerase from influenza C virus.

Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.

Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA.

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3'-5' exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3'-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5'-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Šresolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain.

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. IMPORTANCE: Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.

X-ray structure of the pestivirus NS3 helicase and its conformation in solution.

UNLABELLED: Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. IMPORTANCE: Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the unwinding specificity of pNS3h, which is active only on RNA duplexes. We also show that pNS3h has a highly dynamic behavior--a characteristic probably shared with NS3 helicases from all Flaviviridae members--that could be targeted for drug design by using recent algorithms to specifically block molecular motion. Compounds that lock the enzyme in a single conformation or limit its dynamic range of conformations are indeed likely to block its helicase function.

Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography.

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused widespread outbreaks of debilitating human disease in the past five years. CHIKV invasion of susceptible cells is mediated by two viral glycoproteins, E1 and E2, which carry the main antigenic determinants and form an icosahedral shell at the virion surface. Glycoprotein E2, derived from furin cleavage of the p62 precursor into E3 and E2, is responsible for receptor binding, and E1 for membrane fusion. In the context of a concerted multidisciplinary effort to understand the biology of CHIKV, here we report the crystal structures of the precursor p62-E1 heterodimer and of the mature E3-E2-E1 glycoprotein complexes. The resulting atomic models allow the synthesis of a wealth of genetic, biochemical, immunological and electron microscopy data accumulated over the years on alphaviruses in general. This combination yields a detailed picture of the functional architecture of the 25 MDa alphavirus surface glycoprotein shell. Together with the accompanying report on the structure of the Sindbis virus E2-E1 heterodimer at acidic pH (ref. 3), this work also provides new insight into the acid-triggered conformational change on the virus particle and its inbuilt inhibition mechanism in the immature complex.

Structure of the phage TP901-1 1.8 MDa baseplate suggests an alternative host adhesion mechanism.

Phages of the Caudovirales order possess a tail that recognizes the host and ensures genome delivery upon infection. The X-ray structure of the approximately 1.8 MDa host adsorption device (baseplate) from the lactococcal phage TP901-1 shows that the receptor-binding proteins are pointing in the direction of the host, suggesting that this organelle is in a conformation ready for host adhesion. This result is in marked contrast with the lactococcal phage p2 situation, whose baseplate is known to undergo huge conformational changes in the presence of Ca(2+) to reach its active state. In vivo infection experiments confirmed these structural observations by demonstrating that Ca(2+) ions are required for host adhesion among p2-like phages (936-species) but have no influence on TP901-1-like phages (P335-species). These data suggest that these two families rely on diverse adhesion strategies which may lead to different signaling for genome release.

Continuous mutual improvement of macromolecular structure models in the PDB and of X-ray crystallographic software: the dual role of deposited experimental data.

Accurate crystal structures of macromolecules are of high importance in the biological and biomedical fields. Models of crystal structures in the Protein Data Bank (PDB) are in general of very high quality as deposited. However, methods for obtaining the best model of a macromolecular structure from a given set of experimental X-ray data continue to progress at a rapid pace, making it possible to improve most PDB entries after their deposition by re-analyzing the original deposited data with more recent software. This possibility represents a very significant departure from the situation that prevailed when the PDB was created, when it was envisioned as a cumulative repository of static contents. A radical paradigm shift for the PDB is therefore proposed, away from the static archive model towards a much more dynamic body of continuously improving results in symbiosis with continuously improving methods and software. These simultaneous improvements in methods and final results are made possible by the current deposition of processed crystallographic data (structure-factor amplitudes) and will be supported further by the deposition of raw data (diffraction images). It is argued that it is both desirable and feasible to carry out small-scale and large-scale efforts to make this paradigm shift a reality. Small-scale efforts would focus on optimizing structures that are of interest to specific investigators. Large-scale efforts would undertake a systematic re-optimization of all of the structures in the PDB, or alternatively the redetermination of groups of structures that are either related to or focused on specific questions. All of the resulting structures should be made generally available, along with the precursor entries, with various views of the structures being made available depending on the types of questions that users are interested in answering.

Anionic bipyridyl ligands for applications in metallasupramolecular chemistry.

The facile synthesis of anionic bipyridyl ligands with dinuclear clathrochelate cores is described. These metalloligands can be obtained in high yields by the reactions of M(ClO4 )2 (H2 O)6 (M: Zn, Mn, or Co) with 4-pyridylboronic acid and 2,6-diformyl-4-methylphenol oxime or 2,6-diformyl-4-tert-butylphenol oxime, followed by deprotonation. The ligands are interesting building blocks for metallasupramolecular chemistry, as evidenced by the formation of a Pt-based molecular square and four coordination polymers with 2D or 3D network structures. Competition experiments reveal that the utilization of anionic bipyridyl ligands can result in significantly more stable assemblies.

X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation.

Arenaviruses are important agents of zoonotic disease worldwide. The virions expose a tripartite envelope glycoprotein complex at their surface, formed by the glycoprotein subunits GP1, GP2 and the stable signal peptide. This complex is responsible for binding to target cells and for the subsequent fusion of viral and host-cell membranes for entry. During this process, the acidic environment of the endosome triggers a fusogenic conformational change in the transmembrane GP2 subunit of the complex. We report here the crystal structure of the recombinant GP2 ectodomain of the lymphocytic choriomeningitis virus, the arenavirus type species, at 1.8-Å resolution. The structure shows the characteristic trimeric coiled coil present in class I viral fusion proteins, with a central stutter that allows a close structural alignment with most of the available structures of class I and III viral fusion proteins. The structure further shows a number of intrachain salt bridges stabilizing the postfusion hairpin conformation, one of which involves an aspartic acid that appears released from a critical interaction with the stable signal peptide upon low pH activation.

Self-assembly of a giant molecular Solomon link from 30 subcomponents.

The synthesis of topologically complex structures, such as links and knots, is one of the current challenges in supramolecular chemistry. The so-called Solomon link consists of two doubly interlocked rings. Despite being a rather simple link from a topological point of view, only few molecular versions of this link have been described so far. Here, we report the quantitative synthesis of a giant molecular Solomon link from 30 subcomponents. The highly charged structure is formed by assembly of 12 cis-blocked Pt(2+) complexes, six Cu(+) ions, and 12 rigid N-donor ligands. Each of the two interlocked rings is composed of six repeating Pt(ligand) units, while the six Cu(+) ions connect the two rings. With a molecular weight of nearly 12 kDa and a diameter of 44.2 Å, this complex is the largest non-DNA-based Solomon link described so far. Furthermore, it represents a molecular version of a "stick link".

Advanced exploitation of unmerged reflection data during processing and refinement with autoPROC and BUSTER.

The validation of structural models obtained by macromolecular X-ray crystallography against experimental diffraction data, whether before deposition into the PDB or after, is typically carried out exclusively against the merged data that are eventually archived along with the atomic coordinates. It is shown here that the availability of unmerged reflection data enables valuable additional analyses to be performed that yield improvements in the final models, and tools are presented to implement them, together with examples of the results to which they give access. The first example is the automatic identification and removal of image ranges affected by loss of crystal centering or by excessive decay of the diffraction pattern as a result of radiation damage. The second example is the `reflection-auditing' process, whereby individual merged data items showing especially poor agreement with model predictions during refinement are investigated thanks to the specific metadata (such as image number and detector position) that are available for the corresponding unmerged data, potentially revealing previously undiagnosed instrumental, experimental or processing problems. The third example is the calculation of so-called F(early) - F(late) maps from carefully selected subsets of unmerged amplitude data, which can not only highlight the location and extent of radiation damage but can also provide guidance towards suitable fine-grained parametrizations to model the localized effects of such damage.

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.

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.

Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.

Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2'-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Šresolution, which shows nsp10 bound to nsp16 through a ∼930 Ų surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in +RNA viruses.

Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody.

Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class III viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor binding domain (RBD) of glycoprotein D fused to a truncated version of gH lacking its N-terminal domain. We report here the 2.1-Å resolution structure of the core fragment of gH present in this chimera, bound to the Fab fragment of a PrV gH-specific monoclonal antibody. The structure strongly complements the information derived from the recently reported structure of gH/gL from herpes simplex virus type 2 (HSV-2). Together with the structure of Epstein-Barr virus (EBV) gH/gL reported in parallel, it provides insight into potentially functional conserved structural features. One feature is the presence of a syntaxin motif, and the other is an extended "flap" masking a conserved hydrophobic patch in the C-terminal domain, which is closest to the viral membrane. The negative electrostatic surface potential of this domain suggests repulsive interactions with the lipid heads. The structure indicates the possible unmasking of an extended hydrophobic patch by movement of the flap during a receptor-triggered conformational change of gH, exposing a hydrophobic surface to interact with the viral membrane during the fusion process.

Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic Di-GMP signaling.

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.

Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER.

Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct 'target' structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Šbetween pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.

The N-terminal domain of the arenavirus L protein is an RNA endonuclease essential in mRNA transcription.

Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a 'cap-snatching' mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/ß architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.