Jorge Navaza

Implementation of molecular replacement in AMoRe

An account is given of the molecular replacement method as implemented in the package AMoRe. The overall strategy of the method is presented and the main functions used in the package are described. The most important features of AMoRe are the quality of the fast rotation and translation functions and the facility of multiple inputs to translation and rigid-body refinement functions, which allow for a fast multiple exploration of crystal configurations with a high level of automation.

The Fusion Glycoprotein Shell of Semliki Forest Virus: An Icosahedral Assembly Primed for Fusogenic Activation at Endosomal pH

Semliki Forest virus (SFV) has been extensively studied as a model for analyzing entry of enveloped viruses into target cells. Here we describe the trace of the polypeptide chain of the SFV fusion glycoprotein, E1, derived from an electron density map at 3.5 A resolution and describe its interactions at the surface of the virus. E1 is unexpectedly similar to the flavivirus envelope protein, with three structural domains disposed in the same primary sequence arrangement. These results introduce a new class of membrane fusion proteins which display lateral interactions to induce the necessary curvature and direct budding of closed particles. The resulting surface protein lattice is primed to cause membrane fusion when exposed to the acidic environment of the endosome.

Structure of the outer membrane complex of a type IV secretion system

Type IV secretion systems are secretion nanomachines spanning the two membranes of Gram-negative bacteria. Three proteins, VirB7, VirB9 and VirB10, assemble into a 1.05 megadalton (MDa) core spanning the inner and outer membranes. This core consists of 14 copies of each of the proteins and forms two layers, the I and O layers, inserting in the inner and outer membrane, respectively. Here we present the crystal structure of a ∼0.6 MDa outer-membrane complex containing the entire O layer. This structure is the largest determined for an outer-membrane channel and is unprecedented in being composed of three proteins. Unexpectedly, this structure identifies VirB10 as the outer-membrane channel with a unique hydrophobic double-helical transmembrane region. This structure establishes VirB10 as the only known protein crossing both membranes of Gram-negative bacteria. Comparison of the cryo-electron microscopy (cryo-EM) and crystallographic structures points to conformational changes regulating channel opening and closing.

The Birnavirus Crystal Structure Reveals Structural Relationships Among Icosahedral Viruses.

Double-stranded RNA virions are transcriptionally competent icosahedral particles that must translocate across a lipid bilayer to function within the cytoplasm of the target cell. Birnaviruses are unique among dsRNA viruses as they have a single T = 13 icosahedral shell, lacking the characteristic inner capsid observed in the others. We determined the crystal structures of the T = 1 subviral particle (260 angstroms in diameter) and of the T = 13 intact virus particle (700 angstroms in diameter) of an avian birnavirus to 3 angstroms and 7 angstroms resolution, respectively. Our results show that VP2, the only component of the virus icosahedral capsid, is homologous both to the capsid protein of positive-strand RNA viruses, like the T = 3 nodaviruses, and to the T = 13 capsid protein of members of the Reoviridae family of dsRNA viruses. Together, these results provide important insights into the multiple functions of the birnavirus capsid and reveal unexpected structural relationships among icosahedral viruses.

Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion

The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple‐layered viral capsid. Here we present the crystal structure of VP6 determined to 2 Å resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal β‐barrel domain and a proximal α‐helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3‐fold axis. A distinguishing feature of the VP6 trimer is a central Zn2+ ion located on the 3‐fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi‐equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer.

Domain Organization in Clostridium botulinum Neurotoxin Type E Is Unique: Its Implication in Faster Translocation

Clostridium botulinum produces seven antigenically distinct neurotoxins [C. botulinum neurotoxins (BoNTs) A-G] sharing a significant sequence homology. Based on sequence and functional similarity, it was believed that their three-dimensional structures will also be similar. Indeed, the crystal structures of BoNTs A and B exhibit similar fold and domain association where the translocation domain is flanked on either side by binding and catalytic domains. Here, we report the crystal structure of BoNT E holotoxin and show that the domain association is different and unique, although the individual domains are similar to those of BoNTs A and B. In BoNT E, both the binding domain and the catalytic domain are on the same side of the translocation domain, and all three have mutual interfaces. This unique association may have an effect on the rate of translocation, with the molecule strategically positioned in the vesicle for quick entry into cytosol. Botulism, the disease caused by BoNT E, sets in faster than any other serotype because of its speedy internalization and translocation, and the present structure offers a credible explanation. We propose that the translocation domain in other BoNTs follows a two-step process to attain translocation-competent conformation as in BoNT E. We also suggest that this translocation-competent conformation in BoNT E is a probable reason for its faster toxic rate compared to BoNT A. However, this needs further experimental elucidation.

On the fitting of model electron densities into EM reconstructions: a reciprocal-space formulation

A fast method for fitting model electron densities into EM reconstructions is presented. The methodology was inspired by the molecular-replacement technique, adapted to take into account phase information and the symmetry imposed during the EM reconstruction. Calculations are performed in reciprocal space, which enables the selection of large volumes of the EM maps, thus avoiding the bias introduced when defining the boundaries of the target density.

Visualization of the Target-Membrane-Inserted Fusion Protein of Semliki Forest Virus by Combined Electron Microscopy and Crystallography

Semliki Forest virus enters cells by receptor-mediated endocytosis. The acidic environment of the endosome triggers a membrane fusion reaction that is mediated by the E1 glycoprotein. During fusion, E1 rearranges from an E1/E2 heterodimer to a highly stable, membrane-inserted E1 homotrimer (E1HT). In this study, we analyzed E1HT by a combination of electron cryomicroscopy, electron crystallography of negatively stained 2D crystals, and fitting of the available X-ray structure of the monomeric E1 ectodomain into the resulting 3D reconstruction. The visualized E1HT reveals that the ectodomain has reoriented vertically and inserted the distal tip of domain II into the lipid bilayer. Our data allow the visualization of a viral fusion protein inserted in its target membrane and demonstrate that insertion is a cooperative process, resulting in rings composed of five to six homotrimers.

Structural polymorphism of the major capsid protein of rotavirus

Rotaviruses are important human pathogens with a triple‐layered icosahedral capsid. The major capsid protein VP6 is shown here to self‐assemble into spherical or helical particles mainly depending upon pH. Assembly is inhibited either by low pH (<3.0) or by a high concentration (>100 mM) of divalent cations (Ca2+ and Zn2+). The structures of two types of helical tubes were determined by electron cryomicroscopy and image analysis to a resolution of 2.0 and 2.5 nm. In both reconstructions, the molecular envelope of VP6 fits the atomic model determined by X‐ray crystallography remarkably well. The 3‐fold symmetry of the VP6 trimer, being incompatible with the helical symmetry, is broken at the level of the trimer contacts. One type of contact is maintained within all VP6 particles (tubes and virus), strongly suggesting that VP6 assemblies arise from different packings of a unique dimer of trimers. Our data show that the protonation state and thus the charge distribution are important switches governing the assembly of macromolecular assemblies.

The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry.

Double‐stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 Å X‐ray structure of a picobirnavirus—an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two‐fold symmetric dimers of a coat protein (CP) with a new 3D‐fold. We show that, as many non‐enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post‐translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120‐subunits capsid has evolved animal cell invasion properties.