RESUMEN
When embarking upon X-ray diffraction data collection from a potentially novel macromolecular crystal form, it can be useful to ascertain whether the measured data reflect a crystal form that is already recorded in the Protein Data Bank and, if so, whether it is part of a large family of related structures. Providing such information to crystallographers conveniently and quickly, as soon as the first images have been recorded and the unit cell characterized at an X-ray beamline, has the potential to save time and effort as well as pointing to possible search models for molecular replacement. Given an input unit cell, and optionally a space group, Nearest-cell rapidly scans the Protein Data Bank and retrieves near-matches.
Asunto(s)
Cristalografía por Rayos X , Bases de Datos de Proteínas , Proteínas/química , Algoritmos , Conformación ProteicaRESUMEN
SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.
Asunto(s)
Biología Computacional/estadística & datos numéricos , Proteómica/estadística & datos numéricos , Cristalización , Interpretación Estadística de Datos , Gestión de la Información , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Programas InformáticosRESUMEN
Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His(6)-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore ;expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biosíntesis , Algoritmos , Medios de Cultivo , Vectores Genéticos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reproducibilidad de los Resultados , Solubilidad , TemperaturaRESUMEN
The icosahedral bluetongue virus (BTV) particle (~80 nm diameter) is composed of three distinct protein layers. These include the subcore shell (VP3), core-surface layer (VP7) and outer capsid layer (VP2 and VP5). The core also contains ten dsRNA genome segments and three minor proteins (VP1[Pol], VP4[CaP]and VP6[Hel]), which form transcriptase complexes. The atomic structure of the BTV core has been determined by X-ray crystallography, demonstrating how the major core proteins are assembled and interact. The VP3 subcore shell assembles at an early stage of virus morphogenesis and not only determines the internal organisation of the genome and transcriptase complexes, but also forms a scaffold for assembly of the outer protein layers. The BTV polymerase (VP1) and VP3 have many functional constraints and equivalent proteins have been identified throughout the Reoviridae, and even in some other families of dsRNA viruses. Variations in these highly conserved proteins can be used to identify members of different genera (e.g. by comparing the polymerase) and different virus species (serogroups) within the genus Orbivirus (e.g. by comparison of VP3). This has helped to identify three new genera within the Reoviridae and two new Orbivirus species. In contrast, sequences of the BTV outer capsid proteins (involved in interactions with neutralising antibodies) are much more variable (particularly VP2) and comprehensive sequence analyses for the 24 types demonstrate that they can be used to identify BTV serotype. The 21 species (158 serotypes) currently recognised within the genus Orbivirus are listed, along with 11 unassigned viruses.
RESUMEN
Double-stranded RNA (dsRNA) viruses conceal their genome from the host to avoid triggering unfavorable cellular responses. The crystal structure of the core of one such virus, bluetongue virus, reveals an outer surface festooned with dsRNA. This may represent a deliberate strategy to sequester dsRNA released from damaged particles to prevent host cell shutoff.
Asunto(s)
Virus de la Lengua Azul/metabolismo , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Proteínas del Núcleo Viral/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , ARN Bicatenario/química , ARN Viral/química , Proteínas del Núcleo Viral/química , Difracción de Rayos XRESUMEN
The bluetongue virus core is a molecular machine that simultaneously and repeatedly transcribes mRNA from 10 segments of viral double-stranded RNA, packaged in a liquid crystalline array. To determine how the logistical problems of transcription within a sealed shell are solved, core crystals were soaked with various ligands and analysed by X-ray crystallography. Mg(2+) ions produce a slight expansion of the capsid around the 5-fold axes. Oligonucleotide soaks demonstrate that the 5-fold pore, opened up by this expansion, is the exit site for mRNA, whilst nucleotide soaks pinpoint a separate binding site that appears to be a selective channel for the entry and exit of substrates and by-products. Finally, nucleotides also bind to the outer core layer, providing a substrate sink.
Asunto(s)
Virus de la Lengua Azul/fisiología , Transcripción Genética , Secuencia de Bases , Sitios de Unión , Virus de la Lengua Azul/genética , Calcio/metabolismo , Cristalografía por Rayos X , Magnesio/metabolismo , Fosfatos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sulfatos/metabolismoRESUMEN
Most non-nucleoside reverse transcriptase (RT) inhibitors are specific for HIV-1 RT and demonstrate minimal inhibition of HIV-2 RT. However, we report that members of the phenylethylthiazolylthiourea (PETT) series of non-nucleoside reverse transcriptase inhibitors showing high potency against HIV-1 RT have varying abilities to inhibit HIV-2 RT. Thus, PETT-1 inhibits HIV-1 RT with an IC(50) of 6 nM but shows only weak inhibition of HIV-2 RT, whereas PETT-2 retains similar potency against HIV-1 RT (IC(50) of 5 nM) and also inhibits HIV-2 RT (IC(50) of 2.2 microM). X-ray crystallographic structure determinations of PETT-1 and PETT-2 in complexes with HIV-1 RT reveal the compounds bind in an overall similar conformation albeit with some differences in their interactions with the protein. To investigate whether PETT-2 could be acting at a different site on HIV-2 RT (e.g. the dNTP or template primer binding site), we compared modes of inhibition for PETT-2 against HIV-1 and HIV-2 RT. PETT-2 was a noncompetitive inhibitor with respect to the dGTP substrate for both HIV-1 and HIV-2 RTs. PETT-2 was also a noncompetitive inhibitor with respect to a poly(rC).(dG) template primer for HIV-2 RT. These results are consistent with PETT-2 binding in corresponding pockets in both HIV-1 and HIV-2 RT with amino acid sequence differences in HIV-2 RT affecting the binding of PETT-2 compared with PETT-1.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Sustancias Intercalantes/farmacología , Piridinas/química , ADN Polimerasa Dirigida por ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Tiazoles/farmacología , Tiourea/análogos & derivados , Triazoles/farmacología , Unión Competitiva , Cristalografía por Rayos X , Cartilla de ADN/metabolismo , Nucleótidos de Desoxiguanina/farmacología , Relación Dosis-Respuesta a Droga , Concentración 50 Inhibidora , Cinética , Modelos Químicos , Modelos Moleculares , Unión Proteica , Piridinas/farmacología , Análisis de Regresión , Inhibidores de la Transcriptasa Inversa/química , Tiourea/química , Tiourea/farmacologíaRESUMEN
The concentration of double-stranded RNA within the bluetongue virus core renders the genome segments liquid crystalline. Powder diffraction rings confirm this local ordering with a 30 A separation between strands. Determination of the structure of the bluetongue virus core serotype 10 and comparison with that of serotype 1 reveals most of the genomic double-stranded RNA, packaged as well-ordered layers surrounding putative transcription complexes at the apices of the particle. The outer layer of RNA is sufficiently well ordered by interaction with the capsid that a model can be built and extended to the less-ordered inner layers, providing a structural framework for understanding the mechanism of this complex transcriptional machine. We show that the genome segments maintain local order during transcription.
Asunto(s)
Virus de la Lengua Azul/genética , Conformación de Ácido Nucleico , ARN Bicatenario/química , ARN Viral/química , Animales , Virus de la Lengua Azul/fisiología , Cristalografía por Rayos X , Genoma Viral , Iones , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Transcripción Genética , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/metabolismo , Ensamble de Virus , Difracción de Rayos XRESUMEN
The structure of the core particle of bluetongue virus has been determined by X-ray crystallography at a resolution approaching 3.5 A. This transcriptionally active compartment, 700 A in diameter, represents the largest molecular structure determined in such detail. The atomic structure indicates how approximately 1,000 protein components self-assemble, using both the classical mechanism of quasi-equivalent contacts, which are achieved through triangulation, and a different method, which we term geometrical quasi-equivalence.
Asunto(s)
Virus de la Lengua Azul/química , Proteínas del Núcleo Viral/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , ARN Viral/química , Proteínas del Núcleo Viral/metabolismoRESUMEN
We are using crystallographic methods to investigate the structure of AHSV and BTV. Our initial approach was to investigate the structure of the major protein component of the viral core, VP7(T13). This trimeric protein has been studied in several crystal forms from both orbiviruses and reveals a structure made up of conserved domains, capable of conformational changes and possessing a cleavage site. Further crystallographic analyses of native particles have provided a picture of the VP7(T13) and VP3(T2) layers of the BTV core. The VP7(T13) layer consists of 260 trimers arranged rather symmetrically and possessing very similar structures, thereby following the rules of quasi equivalence. The VP3(T2) layer is thin and contains 120 copies of 100 kDa protein arranged as 60 approximate dimers. This type of icosahedral construction has not been observed before and appears to contain a genome which is highly ordered. We anticipate that all of these features will be common to AHSV.