ISPyB for BioSAXS, the gateway to user autonomy in solution scattering experiments

Antolinos, Alejandro De María; Pernot, Pétra; Brennich, Martha; Kieffer, Jérôme; Bowler, Matthew W.; Delageniere, S.; Ohlsson, S.; Monaco, S.; Ashton, Alun; Franke, Daniel; Svergun, Dmitri I.; McSweeney, Seán; Gordón, E.; Round, Adam

doi:10.1107/s1399004714019609

Cited by 61 publications

(49 citation statements)

References 17 publications

(14 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To obtain the measurements, 30 l of protein solution at three different concentrations for each sample alternated with buffer measurements for background subtraction and the robotic sample handling available at the beamline were used (17). The experiments were programmed using the ISPyB BioSAXS interface (18), and the sequence was triggered via the BsxCuBE control software. Ten individual frames for every exposure, each of which was 2 s in duration, were collected using a Pilatus 1M detector (Dectris).…”

Section: Size Exclusion Chromatography (Sec) Combined With Detection mentioning

confidence: 99%

Domain Organization of Vaccinia Virus Helicase-Primase D5

Hutin

Ling

Round

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae. We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single-and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins. IMPORTANCESince the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design. V iruses of the family Poxviridae have a large linear doublestranded DNA (dsDNA) genome coding for up to 250 open reading frames. The double-stranded DNA genome is covalently circularized at both ends and is fully replicated in the cytoplasm. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) (1) and in the Phycodnaviridae (reviewed in reference 2). Like poxviruses, these viruses are also members of the nucleocytoplasmic large DNA virus (NCLDV) clade (3).We are working on vaccinia virus, which was used for the vaccination campaign that led to the eradication of smallpox. It is a convenient and rather safe system for the study of general poxviru...

show abstract

Section: Size Exclusion Chromatography (Sec) Combined With Detection mentioning

confidence: 99%

Domain Organization of Vaccinia Virus Helicase-Primase D5

Hutin

Ling

Round

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Measurements of 40 ll of BGLF2/3 solution used three different concentrations (1.3, 0.65 and 0.325 mg/ml). Experimental conditions were entered into the ISPyB BioSAXS interface [30] and data were collected via the BsxCuBE using robotic sample handling [31]. Ten individual frames of 1 s exposure were collected using the Pilatus 1 M detector (Dectris).…”

Section: Small-angle X-ray Scatteringmentioning

confidence: 99%

Production and characterisation of Epstein–Barr virus helicase–primase complex and its accessory protein BBLF2/3

et al. 2015

Self Cite

View full text Add to dashboard Cite

The helicase-primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein-Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase-primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase-primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases.

show abstract

“…19. Open "data acquisition" in ISPyB 20 and press "Go" to look at the data set and the automatic analysis 5. Open the damsel.log file in a text editor.…”

Section: Sec-saxs Data Collectionmentioning

confidence: 99%

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Brennich¹,

Round²,

Hutin³

2017

JoVE

Self Cite

View full text Add to dashboard Cite

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC-and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

show abstract

ISPyB for BioSAXS, the gateway to user autonomy in solution scattering experiments

Abstract: The ISPyB information-management system for crystallography has been adapted to include data from small-angle X-ray scattering of macromolecules in solution experiments.

Cited by 61 publications

References 17 publications

Domain Organization of Vaccinia Virus Helicase-Primase D5

Domain Organization of Vaccinia Virus Helicase-Primase D5

Production and characterisation of Epstein–Barr virus helicase–primase complex and its accessory protein BBLF2/3

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Contact Info

Product

Resources

About