Assembly mechanism of the pleomorphic immature poxvirus scaffold.

In Vaccinia virus (VACV), the prototype poxvirus, scaffold protein D13 forms a honeycomb-like lattice on the viral membrane that results in formation of the pleomorphic immature virion (IV). The structure of D13 is similar to those of major capsid proteins that readily form icosahedral capsids in nucleocytoplasmic large DNA viruses (NCLDVs). However, the detailed assembly mechanism of the nonicosahedral poxvirus scaffold has never been understood. Here we show the cryo-EM structures of the D13 trimer and scaffold intermediates produced in vitro. The structures reveal that the displacement of the short N-terminal α-helix is critical for initiation of D13 self-assembly. The continuous curvature of the IV is mediated by electrostatic interactions that induce torsion between trimers. The assembly mechanism explains the semiordered capsid-like arrangement of D13 that is distinct from icosahedral NCLDVs. Our structures explain how a single protein can self-assemble into different capsid morphologies and represent a local exception to the universal Caspar-Klug theory of quasi-equivalence.

Structure of the bacterial flagellar hook cap provides insights into a hook assembly mechanism.

Assembly of bacterial flagellar hook requires FlgD, a protein known to form the hook cap. Symmetry mismatch between the hook and the hook cap is believed to drive efficient assembly of the hook in a way similar to the filament cap helping filament assembly. However, the hook cap dependent mechanism of hook assembly has remained poorly understood. Here, we report the crystal structure of the hook cap composed of five subunits of FlgD from Salmonella enterica at 3.3 Å resolution. The pentameric structure of the hook cap is divided into two parts: a stalk region composed of five N-terminal domains; and a petal region containing five C-terminal domains. Biochemical and genetic analyses show that the N-terminal domains of the hook cap is essential for the hook-capping function, and the structure now clearly reveals why. A plausible hook assembly mechanism promoted by the hook cap is proposed based on the structure.

Structure of polymerized type V pilin reveals assembly mechanism involving protease-mediated strand exchange.

Bacterial adhesion is a general strategy for host-microbe and microbe-microbe interactions. Adhesive pili are essential for colonization, biofilm formation, virulence and pathogenesis of many environmental and pathogenic bacteria1,2. Members of the class Bacteroidia have unique type V pili, assembled by protease-mediated polymerization3. Porphyromonas gingivalis is the main contributor to periodontal disease and its type V pili are a key factor for its virulence4. However, the structure of the polymerized pilus and its assembly mechanism are unknown. Here we show structures of polymerized and monomeric states of FimA stalk pilin from P. gingivalis, determined by cryo-electron microscopy and crystallography. The atomic model of assembled FimA shows that the C-terminal strand of a donor subunit is inserted into a groove in the ß-sheet of an acceptor subunit after N-terminal cleavage by the protease RgpB. The C terminus of the donor strand is essential for polymerization. We propose that type V pili assemble via a sequential polar assembly mechanism at the cell surface, involving protease-mediated strand exchange, employed by various Gram-negative species belonging to the class Bacteroidia. Our results reveal functional surfaces related to pathogenic properties of polymerized FimA. These insights may facilitate development of antibacterial drugs.

Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome.

The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.

Torque transmission mechanism of the curved bacterial flagellar hook revealed by cryo-EM.

Bacterial locomotion by rotating flagella is achieved through the hook, which transmits torque from the motor to the filament. The hook is a tubular structure composed of a single type of protein, yet it adopts a curved shape. To perform its function, it must be simultaneously flexible and torsionally rigid. The molecular mechanism by which chemically identical subunits form such a dynamic structure is unknown. Here, we show the complete structure of the hook from Salmonella enterica in its supercoiled 'curved' state, at 2.9 Å resolution. Subunits in the curved hook are grouped into 11 distinctive conformations, each shared along 11 protofilaments. The domains of the elongated hook subunit behave as rigid bodies connected by two hinge regions. The reconstituted model demonstrates how identical subunits can dynamically change conformation by physical interactions while bending. These multiple subunit states contradict the two-state model, which is a key feature of flagellar polymorphism.

Architecture of the Bacterial Flagellar Distal Rod and Hook of Salmonella.

The bacterial flagellum is a large molecular complex composed of thousands of protein subunits for motility. The filamentous part of the flagellum, which is called the axial structure, consists of the filament, the hook, and the rods, with other minor components-the cap protein and the hook associated proteins. They share a common basic architecture of subunit arrangement, but each part shows quite distinct mechanical properties to achieve its specific function. The distal rod and the hook are helical assemblies of a single protein, FlgG and FlgE, respectively. They show a significant sequence similarity but have distinct mechanical characteristics. The rod is a rigid, straight cylinder, whereas the hook is a curved tube with high bending flexibility. Here, we report a structural model of the rod constructed by using the crystal structure of a core fragment of FlgG with a density map obtained previously by electron cryomicroscopy. Our structural model suggests that a segment called L-stretch plays a key role in achieving the distinct mechanical properties of the rod using a structurally similar component protein to that of the hook.

Metastable asymmetrical structure of a shaftless V1 motor.

V1-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A3B3 complex and a central DF shaft. The nucleotide-free A3B3 complex of Enterococcus hirae, composed of three identical A1B1 heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A3B3. The nucleotide-free mutant A3B3 complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A3B3 forms a metastable (stressed) asymmetric structure composed of unstable A1B1 conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.

Cryo-EM structure of the Ebola virus nucleoprotein-RNA complex at 3.6 Å resolution.

Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical nucleoprotein-RNA complex, VP24, VP30, VP35 and viral polymerase1,3. The nucleoprotein-RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion4,5. RNA binding and nucleoprotein oligomerization are synergistic and do not readily occur independently6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core4,5, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola virus nucleoprotein-RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development.

Evidence for the hook supercoiling mechanism of the bacterial flagellum.

The bacterial flagellar hook is a short, highly curved tubular structure connecting the basal body as a rotary motor and the filament as a helical propeller to function as a universal joint to transmit motor torque to the filament regardless of its orientation. This highly curved form is known to be part of a supercoil as observed in the polyhook structure. The subunit packing interactions in the Salmonella hook structure solved in the straight form gave clear insights into the mechanisms of its bending flexibility and twisting rigidity. Salmonella FlgE consists of four domains, D0, Dc, D1 and D2, arranged from inside to outside of the tube, and an atomic model of the supercoiled hook built to simulate the hook shape observed in the native flagellum suggested that the supercoiled form is stabilized by near-axial interactions of the D2 domains on the inner surface of the supercoil. Here we show that the deletion of domain D2 from FlgE makes the hook straight, providing evidence to support the proposed hook supercoiling mechanism that it is the near-axial interactions between the D2 domains that stabilize the highly curved hook structure.

The FlaG regulator is involved in length control of the polar flagella of Campylobacter jejuni.

Campylobacter jejuni cells have bipolar flagella. Both flagella have similar lengths of about one helical turn, or 3.53±0.52 µm. The flagellar filament is composed of two homologous flagellins: FlaA and FlaB. Mutant strains that express either FlaA or FlaB alone produce filaments that are shorter than those of the wild-type. It is reported that the flaG gene could affect filament length in some species of bacteria, but its function remains unknown. We introduced a flaG-deletion mutation into the C. jejuni wild-type strain and flaA- or flaB-deletion mutant strains, and observed their flagella by microscopy. The ΔflaG mutant cells produced long filaments of two helical turns in the wild-type background. The ΔflaAG double mutant cells produced very short FlaB filaments. On the other hand, ΔflaBG double mutant cells produced long FlaA filaments and their morphology was not helical but straight. Furthermore, FlaG was secreted, and a pulldown assay showed that sigma factor 28 was co-precipitated with purified polyhistidine-tagged FlaG. We conclude that FlaG controls flagella length by negatively regulating FlaA filament assembly and discuss the role of FlaA and FlaB flagellins in C. jejuni flagella formation.

Complete structure of the bacterial flagellar hook reveals extensive set of stabilizing interactions.

The bacterial flagellar hook is a tubular helical structure made by the polymerization of multiple copies of a protein, FlgE. Here we report the structure of the hook from Campylobacter jejuni by cryo-electron microscopy at a resolution of 3.5 Å. On the basis of this structure, we show that the hook is stabilized by intricate inter-molecular interactions between FlgE molecules. Extra domains in FlgE, found only in Campylobacter and in related bacteria, bring more stability and robustness to the hook. Functional experiments suggest that Campylobacter requires an unusually strong hook to swim without its flagella being torn off. This structure reveals details of the quaternary organization of the hook that consists of 11 protofilaments. Previous study of the flagellar filament of Campylobacter by electron microscopy showed its quaternary structure made of seven protofilaments. Therefore, this study puts in evidence the difference between the quaternary structures of a bacterial filament and its hook.

Structural insights into bacterial flagellar hooks similarities and specificities.

Across bacteria, the protein that makes the flagellar hook, FlgE, has a high variability in amino acid residue composition and sequence length. We hereby present the structure of two fragments of FlgE protein from Campylobacter jejuni and from Caulobacter crescentus, which were obtained by X-ray crystallography, and a high-resolution model of the hook from Caulobacter. By comparing these new structures of FlgE proteins, we show that bacterial hook can be divided in two distinct parts. The first part comprises domains that are found in all FlgE proteins and that will make the basic structure of the hook that is common to all flagellated bacteria. The second part, hyper-variable both in size and structure, will be bacteria dependent. To have a better understanding of the C. jejuni hook, we show that a special strain of Salmonella enterica, which was designed to encode a gene of flgE that has the extra domains found in FlgE from C. jejuni, is fully motile. It seems that no matter the size of the hook protein, the hook will always have a structure made of 11 protofilaments.

Structural flexibility of the periplasmic protein, FlgA, regulates flagellar P-ring assembly in Salmonella enterica.

A periplasmic flagellar chaperone protein, FlgA, is required for P-ring assembly in bacterial flagella of taxa such as Salmonella enterica or Escherichia coli. The mechanism of chaperone-mediated P-ring formation is poorly understood. Here we present the open and closed crystal structures of FlgA from Salmonella enterica serovar Typhimurium, grown under different crystallization conditions. An intramolecular disulfide cross-linked form of FlgA caused a dominant negative effect on motility of the wild-type strain. Pull-down experiments support a specific protein-protein interaction between FlgI, the P-ring component protein, and the C-terminal domain of FlgA. Surface plasmon resonance and limited-proteolysis indicate that flexibility of the domain is reduced in the covalently closed form. These results show that the structural flexibility of the C-terminal domain of FlgA, which is related to the structural difference between the two crystal forms, is intrinsically associated with its molecular chaperone function in P-ring assembly.

Purification, crystallization and preliminary X-ray crystallographic analysis of the flagellar accessory protein FlaH from the methanogenic archaeon Methanocaldococcus jannaschii.

The flagellar accessory protein FlaH is thought to be one of the essential components of an archaeal motility system. However, to date biochemical and structural information about this protein has been limited. Here, the crystallization of FlaH from the hyperthermophilic archaeon Methanocaldococcus jannaschii is reported. Protein crystals were obtained by the vapour-diffusion method. These crystals belonged to space group P3121, with unit-cell parameters a=b=131.42, c=89.35 Å. The initial solution of the FlaH structure has been determined by multiple-wavelength anomalous dispersion phasing using a selenomethionine-derivatized crystal.

Crystallization and preliminary X-ray diffraction analysis of the periplasmic domain of the Escherichia coli aspartate receptor Tar and its complex with aspartate.

The cell-surface receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). To understand the molecular mechanisms underlying the induction of Tar activity by its ligands, the Escherichia coli Tar periplasmic domain with and without bound aspartate (Asp-Tar and apo-Tar, respectively) were each crystallized in two different forms. Using ammonium sulfate as a precipitant, crystals of apo-Tar1 and Asp-Tar1 were grown and diffracted to resolutions of 2.10 and 2.40 Å, respectively. Alternatively, using sodium chloride as a precipitant, crystals of apo-Tar2 and Asp-Tar2 were grown and diffracted to resolutions of 1.95 and 1.58 Å, respectively. Crystals of apo-Tar1 and Asp-Tar1 adopted space group P41212, while those of apo-Tar2 and Asp-Tar2 adopted space groups P212121 and C2, respectively.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a core fragment of FlgG, a bacterial flagellar rod protein.

FlgG is a bacterial flagellar rod protein and constructs the distal rod connecting to the hook. FlgG of Salmonella enterica serovar Typhimurium is a 260-amino-acid protein composed of a folded core region and N- and C-terminal regions that are unfolded in solution. A core fragment of FlgG (FlgG47-227) was expressed, purified and crystallized. Crystals of native and SeMet-labelled FlgG47-227 were obtained by the sitting-drop vapour-diffusion technique with PEG MME 2000 as precipitant. The native crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 47.78, b = 68.94, c = 110.57 Å. The SeMet crystal also belonged to space group P212121, with unit-cell parameters a = 47.53, b = 67.04, c = 110.27 Å.

Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins.

The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBC). Here, the crystal structures of FlhBC from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhBC structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhBC leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhBC and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhBC molecule.

Crystallization and preliminary X-ray analysis of FlgA, a periplasmic protein essential for flagellar P-ring assembly.

Salmonella FlgA, a periplasmic protein essential for flagellar P-ring assembly, has been crystallized in two forms. The native protein crystallized in space group C222, with unit-cell parameters a = 107.5, b = 131.8, c = 49.4 Å, and diffracted to about 2.0 Å resolution (crystal form I). In this crystal, the asymmetric unit is likely to contain one molecule, with a solvent content of 66.8%. Selenomethionine-labelled FlgA protein crystallized in space group C222(1), with unit-cell parameters a = 53.2, b = 162.5, c = 103.5 Å, and diffracted to 2.7 Å resolution (crystal form II). In crystal form II, the asymmetric unit contained two molecules with a solvent content of 48.0%. The multiple-wavelength and single-wavelength anomalous dispersion methods allowed the visualization of the electron-density distributions of the form I and II crystals, respectively. The two maps suggested that FlgA is in two different conformations in the two crystals.

Purification, crystallization and preliminary X-ray analysis of FliT, a bacterial flagellar substrate-specific export chaperone.

The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD(4)C(2) complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3(1)21 or P3(2)21 and diffracted to 3.2 A resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination.