Delivery of the rhinovirus genome into the cytoplasm involves a cooperative structural modification of the viral capsid. We have studied this phenomenon for human rhinovirus serotype 2 (HRV2). The ...structure of the empty capsid has been determined to a resolution of better than 15 Å by cryo-electron microscopy, and the atomic structure of native HRV2 was used to examine conformational changes of the capsid. The two proteins around the 5-fold axes make an iris type of movement to open a 10 Å diameter channel which allows the RNA genome to exit, and the N terminus of VP1 exits the capsid at the pseudo 3-fold axis. A remarkable modification occurs at the 2-fold axes where the N-terminal loop of VP2 bends inward, probably to detach the RNA.
Architecture of the VE-cadherin Hexamer Hewat, Elizabeth A.; Durmort, Claire; Jacquamet, Lilian ...
Journal of molecular biology,
01/2007, Letnik:
365, Številka:
3
Journal Article
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Vascular endothelial-cadherin (VE-cadherin) is the major constituent of the adherens junctions of endothelial cells and plays a key role in angiogenesis and vascular permeability. The ectodomains ...EC1-4 of VE-cadherin are known to form hexamers in solution. To examine the mechanism of homotypic association of VE-cadherin, we have made a 3D reconstruction of the EC1-4 hexamer using electron microscopy and produced a homology model based on the known structure of C-cadherin EC1-5. The hexamer consists of a trimer of dimers with each N-terminal EC1 module making an antiparallel dimeric contact, and the EC4 modules forming extensive trimeric interactions. Each EC1-4 molecule makes a helical curve allowing some torsional flexibility to the edifice. While there is no direct evidence for the existence of hexamers of cadherin at adherens junctions, the model that we have produced provides indirect evidence since it can be used to explain some of the disparate results for adherens junctions. It is in accord with the X-ray and electron microscopy results, which demonstrate that the EC1 dimer is central to homotypic cadherin interaction. It provides an explanation for the force measurements of the interaction between opposing cadherin layers, which have previously been interpreted as resulting from three different interdigitating interactions. It is in accord with observations of native junctions by cryo-electron microscopy. The fact that this hexameric model of VE-cadherin can be used to explain more of the existing data on adherens junctions than any other model alone argues in favour of the existence of the hexamer at the adherens junction. In the context of the cell—cell junction these cis-trimers close to the membrane, and trans-dimers from opposing membranes, would increase the avidity of the bond.
Junonia coenia densovirus (JcDNV) belongs to the densovirus genus of the Parvoviridae family and infects the larvae of the Common Buckeye butterfly. Its capsid is icosahedral and consists of viral ...proteins VP1 (88
kDa), VP2 (58
kDa), VP3 (52
kDa) and VP4 (47
kDa). Each viral protein has the same C terminus but differs in the length of its N-terminal extension. Virus-like-particles (VLPs) assemble spontaneously when the individual viral proteins are expressed by a recombinant baculovirus. We present here the structure of native JcDNV at 8.7
Å resolution and of the two VLPs formed essentially from VP2 and VP4 at 17
Å resolution, as determined by cryo-electron microscopy. The capsid displays a remarkably smooth surface, with only two very small spikes that define a pentagonal plateau on the 5-fold axes. JcDNV is very closely related to
Galleria mellonella densovirus (GmDNV), whose structure is known (94% sequence identity with VP4 and 96% similarity). We compare these structures in order to locate the structural changes and mutations that may be involved in the species shift of these densoviruses. A single mutation at the tip of one of the two small spikes is a strong candidate as a species shift determinant. Difference imaging reveals that the 21 disordered amino acid residues at the N terminus of the capsid protein VP4 are located inside the capsid at the 5-fold axis, but the additional 94 amino acid residue extension of VP2 is not visible, suggesting that it is highly disordered. There is strong evidence of DNA ordering associated with the 3-fold axes of the capsid.
The structure of a complex between rabbit haemorrhagic disease virus (RHDV) virus-like particles (VLPs) and a neutralising monoclonal antibody mAb-E3 has been determined at low resolution by ...cryo-electron microscopy and three-dimensional (3-D) reconstruction techniques. The atomic co-ordinates of an Fab were fitted to the cryo-electron microscope density map to produce a binding model. The VLP has a T = 3 icosahedral lattice consisting of a hollow spherical shell with 90 protruding arches. Each dimeric arch presents two mAb binding sites; however, steric hindrance between the variable domains of the Fabs prevents the occupation of both sites simultaneously. Thus the maximum mAb occupation is 50%. Once a mAb is bound to one site it may bind to either of two neighbouring sites related by a local 3-fold axis. The mAbs are bound bivalently on epitopes not related by a 2-fold symmetry axis. This binding geometry implies a torsional flexibility of the mAb hinge region, involving a 60° rotation of one Fab arm with respect to the other. Owing to extreme flexibility of the hinge region, the Fc domains occupy random orientations and are not visible in the reconstruction. The bivalent attachment of mAb-E3 to RHDV suggests that the neutralisation mechanism(s) involves inhibition of viral decapsidation and/or the inhibition of binding to the receptor.
Data from cryo‐electron microscopy and X‐ray crystallography have been combined to study the interactions of foot‐and‐mouth disease virus serotype C (FMDV‐C) with a strongly neutralizing monoclonal ...antibody (mAb) SD6. The mAb SD6 binds to the long flexible GH‐loop of viral protein 1 (VP1) which also binds to an integrin receptor. The structure of the virus–Fab complex was determined to 30 Å resolution using cryo‐electron microscopy and image analysis. The known structure of FMDV‐C, and of the SD6 Fab co‐crystallized with a synthetic peptide corresponding to the GH‐loop of VP1, were fitted to the cryo‐electron microscope density map. The SD6 Fab is seen to project almost radially from the viral surface in an orientation which is only compatible with monovalent binding of the mAb. Even taking into account the mAb hinge and elbow flexibility, it is not possible to model bivalent binding without severely distorting the Fabs. The bound GH‐loop is essentially in what has previously been termed the ‘up’ position in the best fit Fab orientation. The SD6 Fab interacts almost exclusively with the GH‐loop of VP1, making very few other contacts with the viral capsid. The position and orientation of the SD6 Fab bound to FMDV‐C is in accord with previous immunogenic data.
The value of an electron microscope equipped with a field emission gun (FEG) was first revealed in materials science applications. More recently, the FEG has played a crucial role in breaking the 10Å ...barrier in single-particle reconstructions of frozen hydrated biological molecules. The standard high-resolution performance tests for electron microscopes are made close to focus, at several hundreds of Å underfocus at a magnification of
500
000×
or more. While this is appropriate for materials science specimens, it is not suitable for observing frozen hydrated biological specimens with which the optimum underfocus is of the order of 1 micron or so and the magnification is limited by radiation damage to roughly 30
000 to
60
000×
. Thus, in order to access the performance of a cryo-electron microscope for high-resolution 3D electron microscopy of biological molecules, additional tests are necessary. We present here resolution tests of a 200-kV FEG using frozen hydrated virus suspensions. The extent and amplitude of the contrast transfer function are used as a test of the performance. We propose that small spherical viruses close to 300
Å in diameter, such as the picornaviruses or phages, make good specimens for testing the performance of an electron microscope in cryo-mode.
Two-dimensional crystals of the histidine-tagged-HupR protein, a transcriptional regulator from the photosynthetic bacterium
Rhodobacter capsulatus, were obtained upon specific interaction with a Ni
...2+-chelated lipid monolayer. HupR is a response regulator of the NtrC family; it activates the transcription of the structural genes,
hupSLC, of the NiFehydrogenase. The lipid (Ni-NTA-DOGA) uses the metal chelator nitrilotriacetic group as the hydrophilic headgroup and contains unsaturated oleyl tails to provide the fluidity necessary for two-dimensional protein crystallization. A projection map of the full-length protein at 18 Å resolution was generated by analysing electron microscopy micrographs of negatively stained crystals. The HupR protein appeared to be dimeric and revealed a characteristic “propeller-like” motif. Each monomer forms an L-shaped structure.
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