Generation and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev.

Rev is a key regulatory protein of human immunodeficiency virus type 1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA, thereby allowing the synthesis of structural proteins. Despite its evident importance, the structure of Rev has remained unknown, primarily because Rev's proclivity for polymerization and aggregation is an impediment to crystallization.

Learning about patient safety: organizational context and culture in the education of health care professionals.

Objectives: This study investigated the formal and informal ways pre-registration students from medicine, nursing, physiotherapy and pharmacy learn about keeping patients safe. This paper gives an overview of the study and explores findings in relation to organizational context and culture.

Methods: The study employed a phased design using multiple qualitative methods.

Beta arcades: recurring motifs in naturally occurring and disease-related amyloid fibrils.

Amyloid fibrils are filamentous protein aggregates that accumulate in diseases such as Alzheimer's or type II diabetes. The amyloid-forming protein is disease specific. Amyloids may also be formed in vitro from many other proteins, after first denaturing them.

Influences on self-evaluation during a clinical skills programme for nurses.

Education has moved from teacher to student-centred practices. Increasing emphasis is placed on 'life-long' learning in the context of a rapidly changing knowledge base. Self-evaluation is seen as one strategy to facilitate student-centred continuous professional development.

Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex.

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry.

Structure and energetics of encapsidated DNA in bacteriophage HK97 studied by scanning calorimetry and cryo-electron microscopy.

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis.

Poliovirus 2C protein forms homo-oligomeric structures required for ATPase activity.

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily.

Electron microscopic evidence in support of alpha-solenoid models of proteasomal subunits Rpn1 and Rpn2.

Rpn1 (109 kDa) and Rpn2 (104 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal alpha-solenoid folds composed of 9-11 proteasome/cyclosome repeats, each approximately 40 residues long and containing two alpha-helices and turns [A. V.

Visualization of a missing link in retrovirus capsid assembly.

For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed.

Irregular and Semi-Regular Polyhedral Models for Rous Sarcoma Virus Cores.

Whereas many viruses have capsids of uniquely defined sizes that observe icosahedral symmetry, retrovirus capsids are highly polymorphic. Nevertheless, they may also be described as polyhedral foldings of a fullerene lattice on which the capsid protein (CA) is arrayed. Lacking the high order of symmetry that facilitates the reconstruction of icosahedral capsids from cryo-electron micrographs, the three-dimensional structures of individual retrovirus capsids may be determined by cryo-electron tomography, albeit at lower resolution.

Field-caught permethrin-resistant Anopheles gambiae overexpress CYP6P3, a P450 that metabolises pyrethroids.

Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides.

Mentoring for NHS doctors: perceived benefits across the personal-professional interface.

Objective: To investigate NHS doctors' perceived benefits of being involved in mentoring schemes and to explore the overlaps and relationships between areas of benefit.

Design: Extended qualitative analysis of a multi-site interview study following an interpretivist approach.

Setting: Six NHS mentoring schemes across England.

Virus capsid expansion driven by the capture of mobile surface loops.

The capsids of tailed-DNA bacteriophages first assemble as procapsids, which mature by converting into a new form that is strong enough to contain a densely packed viral chromosome. We demonstrate that the intersubunit crosslinking that occurs during maturation of HK97 capsids actually promotes the structural transformation. Small-angle X-ray scattering and crosslinking assays reveal that a shift in the crosslink pattern accompanies conversion of a semimature particle, Expansion Intermediate-I/II, to a more mature state, Balloon.

The state of the art.

Graphical representation of molecular and cellular features is a key form of communication in structural biology in which abstract symbols of an economical and visually appropriate kind, pseudo-color coding, and dynamic animations all play their parts. Accordingly, it should not be surprising that many structural biologists--like traditional biologists before them--are talented artists who also express themselves on "non-scientific" topics. This article illustrates the approaches of and pictures by several practicing scientist-artists-mainly, in this sampling, electron microscopists.

The future is hybrid.

On the occasion of the 50th anniversary of the Journal of Structural Biology, we review some of the major advances that have taken place in molecular and cellular structural biology over this timeframe and consider some current trends, as well as promising new directions. While the primary experimental techniques of X-ray diffraction, electron microscopy and NMR spectroscopy continue to improve and other powerful new techniques have come on-line, it appears that the most comprehensive analyses of large, dynamic, macromolecular machines will rely on integrated combinations of different methodologies, viz. "hybrid approaches".

High-resolution mass spectrometry of viral assemblies: molecular composition and stability of dimorphic hepatitis B virus capsids.

Hepatitis B virus (HBV) is a major human pathogen. In addition to its importance in human health, there is growing interest in adapting HBV and other viruses for drug delivery and other nanotechnological applications. In both contexts, precise biophysical characterization of these large macromolecular particles is fundamental.

Non-canonical binding of an antibody resembling a naïve B cell receptor immunoglobulin to hepatitis B virus capsids.

The hepatitis B virus capsid (core antigen) is able to bind to and activate naïve B cells and these become efficient primary antigen-presenting cells for the priming of T cells. We have investigated this interaction by using cryo-electron microscopy, three-dimensional image reconstruction, and molecular modeling to visualize capsids decorated with Fab fragments of a receptor immunoglobulin, and surface plasmon resonance to measure the binding affinity. By both criteria, the mode of binding differs from those of the six monoclonal anti-core antigen antibodies previously characterized.

Electron microscopy and image processing: an essential tool for structural analysis of macromolecules.

Macromolecular electron microscopy (EM) deals with macromolecular complexes and their placement within the cell-linking the molecular and cellular worlds as a bridge between atomic-resolution X-ray crystallographic or NMR studies and lower resolution light microscopy. The amount of specimen required is typically 10(2) to 10(3) times less than for X-ray crystallography or NMR. Electron micrographs of frozen-hydrated specimens portray native structures.

Arrangement of L2 within the papillomavirus capsid.

Papillomaviruses are a family of nonenveloped DNA tumor viruses. Some sexually transmitted human papillomavirus (HPV) types, including HPV type 16 (HPV16), cause cancer of the uterine cervix. Papillomaviruses encode two capsid proteins, L1 and L2.

Initial location of the RNA-dependent RNA polymerase in the bacteriophage Phi6 procapsid determined by cryo-electron microscopy.

The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage Phi6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as single-stranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route.

RSV capsid polymorphism correlates with polymerization efficiency and envelope glycoprotein content: implications that nucleation controls morphogenesis.

We used cryo-electron tomography to visualize Rous sarcoma virus, the prototypic alpharetrovirus. Its polyprotein Gag assembles into spherical procapsids, concomitant with budding. In maturation, Gag is dissected into its matrix, capsid protein (CA), and nucleocapsid moieties.

Characterization of beta-sheet structure in Ure2p1-89 yeast prion fibrils by solid-state nuclear magnetic resonance.

Residues 1-89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole-dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N-13C and 13C-13C NMR spectra of samples that are uniformly 15N- and 13C-labeled.