RESUMEN
Contemporary biology is currently undergoing a revolution, driven by the availability of high-throughput technologies and a wide variety of bioinformatics tools. However, bioinformatics education and practice is still in its infancy in most of the African continent. Consequently, concerted efforts have been made in recent years to incorporate bioinformatics modules into biological sciences curriculum of African Universities. Despite this, one aspect of bioinformatics that is yet to be incorporated is structural bioinformatics. In this article, we report on a structural bioinformatics project carried out by final year project students in a Nigerian university. The target protein was the thermoacidophilic Sulfolobus islandicus rod-shaped virus 1 (SIRV1) Rep protein, which was further characterized using various free, user-friendly and online sequence-based and structure-based bioinformatics tools. This exercise gave students the opportunity to generate new data, interpret the data, and acquire collaborative research skills. In this report, emphasis is placed on analysis of the data generated to further encourage analytical skills. By sharing this experience, it is anticipated that other similar institutions would adopt parallel strategies to expose undergraduate students to structural biology, and increase awareness of freely available bioinformatics tools for tackling pertinent biological questions. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):547-554, 2018.
Asunto(s)
Biología Computacional/educación , Rudiviridae/química , Estudiantes , UniversidadesRESUMEN
Viruses infecting hyperthermophilic archaea typically do not encode DNA polymerases, raising questions regarding their genome replication. Here, using a yeast two-hybrid approach, we have assessed interactions between proteins of Sulfolobus islandicus rod-shaped virus 2 (SIRV2) and the host-encoded proliferating cell nuclear antigen (PCNA), a key DNA replication protein in archaea. Five SIRV2 proteins were found to interact with PCNA, providing insights into the recruitment of host replisome for viral DNA replication.
Asunto(s)
Proteínas Arqueales/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Rudiviridae/metabolismo , Sulfolobus/metabolismo , Sulfolobus/virología , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , ADN de Archaea/genética , ADN de Archaea/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Antígeno Nuclear de Célula en Proliferación/genética , Unión Proteica , Rudiviridae/química , Rudiviridae/genética , Sulfolobus/genética , Proteínas Virales/química , Proteínas Virales/genética , Replicación ViralRESUMEN
Single-particle electron cryomicroscopy is undergoing a technical revolution due to the recent developments of direct detectors. These new recording devices detect electrons directly (i.e. without conversion into light) and feature significantly improved detective quantum efficiencies and readout rates as compared to photographic films or CCDs. We evaluated here the potential of one such detector (Gatan K2 Summit) to enable the achievement of near-atomic resolution reconstructions of biological specimens when coupled to a widely used, mid-range transmission electron microscope (FEI TF20 Twin). Compensating for beam-induced motion and stage drift provided a 4.4Å resolution map of Sulfolobus turreted icosahedral virus (STIV), which we used as a test particle in this study. Several motion correction and dose fractionation procedures were explored and we describe their influence on the resolution of the final reconstruction. We also compared the quality of this data to that collected with a FEI Titan Krios microscope equipped with a Falcon I direct detector, which provides a benchmark for data collected using a high-end electron microscope.
Asunto(s)
Microscopía por Crioelectrón/instrumentación , Microscopía Electrónica de Transmisión/instrumentación , Rudiviridae/ultraestructura , Modelos Moleculares , Rudiviridae/química , Sulfolobus/virología , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/ultraestructura , Virión/química , Virión/ultraestructuraRESUMEN
Sulfolobus turreted icosahedral virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of transmembrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses, suggesting that they derive from a common ancestor predating the divergence of the three kingdoms of life. One architectural motif (ß-jelly roll fold) forms virtually the entire capsid (distributed in three different gene products), indicating that a single ancestral protein module may have been at the origin of its evolution.
Asunto(s)
Proteínas de la Membrana/química , Modelos Moleculares , Rudiviridae/química , Sulfolobus/virología , Microscopía por Crioelectrón , Cristalografía por Rayos XRESUMEN
Viruses utilize a diverse array of mechanisms to deliver their genomes into hosts. While great strides have been made in understanding the genome delivery of eukaryotic and prokaryotic viruses, little is known about archaeal virus genome delivery and the associated particle changes. The Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA (dsDNA) archaeal virus that contains a host-derived membrane sandwiched between the genome and the proteinaceous capsid shell. Using cryo-electron microscopy (cryo-EM) and different biochemical treatments, we identified three viral morphologies that may correspond to biochemical disassembly states of STIV. One of these morphologies was subtly different from the previously published 27-A-resolution electron density that was interpreted with the crystal structure of the major capsid protein (MCP). However, these particles could be analyzed at 12.5-A resolution by cryo-EM. Comparing these two structures, we identified the location of multiple proteins forming the large turret-like appendages at the icosahedral vertices, observed heterogeneous glycosylation of the capsid shell, and identified mobile MCP C-terminal arms responsible for tethering and releasing the underlying viral membrane to and from the capsid shell. Collectively, our studies allow us to propose a fusogenic mechanism of genome delivery by STIV, in which the dismantled capsid shell allows for the fusion of the viral and host membranes and the internalization of the viral genome.
Asunto(s)
Rudiviridae/química , Rudiviridae/ultraestructura , Sulfolobus/virología , Virión/química , Virión/ultraestructura , Microscopía por Crioelectrón , Sustancias Macromoleculares , Modelos Biológicos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Proteínas Virales/química , Internalización del VirusRESUMEN
NMR spectroscopy and X-ray crystallography are currently the two most widely applied methods for the determination of macromolecular structures at high resolution. More recently, significant advances have been made in algorithms for the de novo prediction of protein structure, and, in favorable cases, the predicted models agree extremely well with experimentally determined structures. Here, we demonstrate a synergistic combination of NMR spectroscopy, de novo structure prediction, and X-ray crystallography in an effective overall strategy for rapidly determining the structure of the coat protein C-terminal domain from the Sulfolobus islandicus rod-shaped virus (SIRV). This approach takes advantage of the most accessible aspects of each structural technique and may be widely applicable for structure determination.
Asunto(s)
Proteínas de la Cápside/química , Biología Computacional/métodos , Cristalografía por Rayos X/métodos , Rudiviridae/química , Proteínas Virales/química , Algoritmos , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de la Cápside/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Homología de Secuencia de Aminoácido , Sulfolobus/virología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The Holliday junction (or four-way junction) is the universal DNA intermediate whose interaction with resolving proteins is one of the major events in the recombinational process. These proteins, called DNA junction-resolving enzymes or resolvases, bind to the junction and catalyse DNA cleavage, promoting the release of two DNA duplexes. SIRV2 Hjc, a viral resolvase infecting a thermophylic archaeon, has been cloned, expressed and purified. Crystals have been obtained in space group C2, with unit-cell parameters a = 147.8, b = 99.9, c = 87.6, beta = 109.46 degrees, and a full data set has been collected at 3.4 A resolution. The self-rotation function indicates the presence of two dimers in the asymmetric unit and a high solvent content (77%). Molecular-replacement trials using known similar resolvase structures have so far been unsuccessful, indicating possible significant structural rearrangements.
Asunto(s)
Virus de Archaea/enzimología , Resolvasas de Unión Holliday/química , Resolvasas de Unión Holliday/aislamiento & purificación , Rudiviridae/química , Secuencia de Aminoácidos , Virus de Archaea/metabolismo , Secuencia de Bases , Cristalización , Escherichia coli/genética , Escherichia coli/metabolismo , Resolvasas de Unión Holliday/genética , Datos de Secuencia Molecular , Rudiviridae/genética , Rudiviridae/aislamiento & purificación , Rudiviridae/metabolismo , Alineación de Secuencia , Difracción de Rayos XRESUMEN
Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group.