An integrated pipeline for sample preparation and characterization at the EMBL@PETRA3 synchrotron facilities

Boivin, Stéphane; Kozak, Sandra; Rasmussen, Gry; Nemtanu, Ioana Maria; Vieira, Vanessa; Meijers, Rob

doi:10.1016/j.ymeth.2015.08.003

Cited by 16 publications

(7 citation statements)

References 24 publications

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“…Synchrotron SEC-SAXS requires transportation of samples to the beamline, with inherent challenges related to the sensitivity of biological samples to temperature differences, mechanical stress and time. Particularly short-lived perishable samples might even necessitate preparation on site, thus requiring dedicated facilities at the synchrotron (Boivin et al, 2016). In conjunction with the time pressure associated with synchrotron experiments, necessary extensive sample handling on site increases the risk of compromising the experiments.…”

Section: Discussionmentioning

confidence: 99%

Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

et al. 2018

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Section: Discussionmentioning

confidence: 99%

Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

et al. 2018

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“…ITC measurements were performed at 20 °C using a VP-ITC calorimeter at the sample preparation and characterization (SPC) facility of the EMBL in Hamburg (32) with a sample cell containing 1.43 ml of 100–160 μ m of CCHFV WT or E656A mutant endonuclease protein or 140 μ m of LCMV WT protein. Titrations were made by injecting 15 μl of 5–8 m m manganese solution.…”

Section: Methodsmentioning

confidence: 99%

Biochemical and structural studies reveal differences and commonalities among cap-snatching endonucleases from segmented negative-strand RNA viruses

Holm

Kopicki

Busch

et al. 2018

Journal of Biological Chemistry

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Viruses rely on many host cell processes, including the cellular transcription machinery. Segmented negative-strand RNA viruses (sNSV) in particular cannot synthesize the 5′-cap structure for their mRNA but cleave off cellular caps and use the resulting oligonucleotides as primers for their transcription. This cap-snatching mechanism, involving a viral cap-binding site and RNA endonuclease, is both virus-specific and essential for viral proliferation and therefore represents an attractive drug target. Here, we present biochemical and structural results on the putative cap-snatching endonuclease of Crimean–Congo hemorrhagic fever virus (CCHFV), a highly pathogenic bunyavirus belonging to the Nairoviridae family, and of two additional nairoviruses, Erve virus (EREV) and Nairobi sheep disease virus (NSDV). Our findings are presented in the context of other cap-snatching endonucleases, such as the enzymatically active endonuclease from Rift Valley fever virus (RVFV), from Arenaviridae and Bunyavirales, belonging to the His− and His+ endonucleases, respectively, according to the absence or presence of a metal ion–coordinating histidine in the active site. Mutational and metal-binding experiments revealed the presence of only acidic metal-coordinating residues in the active site of the CCHFV domain and a unique active-site conformation that was intermediate between those of His+ and His− endonucleases. On the basis of small-angle X-ray scattering (SAXS) and homology modeling results, we propose a protein topology for the CCHFV domain that, despite its larger size, has a structure overall similar to those of related endonucleases. These results suggest structural and functional conservation of the cap-snatching mechanism among sNSVs.

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“…The interface generates a worksheet with (1) a screen layout comprising a detailed well composition, (2) a table with all components required, comprising a calculation spreadsheet, and (3) a pipetting protocol for each well. The pipetting protocol can be formatted into scripts for a liquid handling robot, such as a Scorpion™ (Art Robbins Instruments) at the Sample Preparation and Characterization (SPC) facility located at the EMBL@PETRA3 synchrotron (Boivin et al 2016 ) ( http://www.embl-hamburg.de/services/spc ). A library of stock solutions was prepared and each solution was filtered before use.…”

Section: Methodsmentioning

confidence: 99%

Optimization of protein samples for NMR using thermal shift assays

et al. 2016

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Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor® provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies. Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-016-0027-z) contains supplementary material, which is available to authorized users.

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An integrated pipeline for sample preparation and characterization at the EMBL@PETRA3 synchrotron facilities

Cited by 16 publications

References 24 publications

Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

Biochemical and structural studies reveal differences and commonalities among cap-snatching endonucleases from segmented negative-strand RNA viruses

Optimization of protein samples for NMR using thermal shift assays

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