A Structural Model of the Genome Packaging Process in a Membrane-Containing Double Stranded DNA Virus

Peer reviewe

Zernike Phase Contrast Cryo-Electron Microscopy and Tomography for Structure Determination at Nanometer and Subnanometer Resolutions

Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of ~3, compared with conventional cryoEM. The reconstruction was carried out to 13 Å resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.National Institutes of Health (U.S.) (Grant P41RR002250)National Institutes of Health (U.S.) (Grant R01AI0175208)National Institutes of Health (U.S.) (Grant PN1EY016525)Robert Welch Foundation (Q1242

In situ structure and assembly of the multidrug efflux pump AcrAB-TolC

Abstract: Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors

Accurate model annotation of a near-atomic resolution cryo-EM map

Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo- EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly.With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.National Institutes of Health (U.S.) (Grant P41GM103832)National Institutes of Health (U.S.) (Grant R01GM079429)National Institutes of Health (U.S.) (Grant PN2EY016525)National Institutes of Health (U.S.) (Grant P01GM063210)Robert A. Welch Foundation (Grant Q1242

Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle

Chaperonins are multisubunit entities that are composed of two stacked rings enclosing a central chamber for ATP-dependent protein folding. A series of cryo-EM structures of the eukaryotic group II chaperonin TRiC/CCT reveal the conformational changes during the ATPase cycle and provide insight into how the subunits cooperate to close the lid

4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

This study uses high-resolution cryo-electron microscopy to provide a complete structural model of the VEEV alphavirus, bridging the gap between incomplete crystal structures and lower resolution electron microscopy analyses

Roles of Triplex and Scaffolding Proteins in Herpes Simplex Virus Type 1 Capsid Formation Suggested by Structures of Recombinant Particles

Typical herpes simplex virus (HSV) capsids contain seven proteins that form a T=16 icosahedron of 1,250-Å diameter. Infection of cells with recombinant baculoviruses expressing two of these proteins, VP5 (which forms the pentons and hexons in typical HSV capsids) and VP19C (a component of the triplexes that connect adjacent capsomeres), results in the formation of spherical particles of 880-Å diameter. Electron cryomicroscopy and computer reconstruction revealed that these particles possess a T=7 icosahedral symmetry, having 12 pentons and 60 hexons. Among the characteristic structural features of the particle are the skewed appearance of the hexons and the presence of intercapsomeric mass densities connecting the middle domain of one hexon subunit to the lower domain of a subunit in the adjacent hexon. We interpret these connecting masses as being formed by VP19C. Comparison of the connecting masses with the triplexes, which occupy equivalent positions in the T=16 capsid, reveals the probable locations of the single VP19C and two VP23 molecules that make up the triplex. Their arrangement suggests that the two triplex proteins have different roles in controlling intercapsomeric interactions and capsid stability. The nature of these particles and of other aberrant forms made in the absence of scaffold demonstrates the conformational adaptability of the capsid proteins and illustrates how VP23 and the scaffolding protein modulate the nature of the VP5-VP19C network to ensure assembly of the functional T=16 capsid