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Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at the Diamond Light Source Beamline I23 by means of S-SAD

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Date
01/11/2016
Author
Bent, Andrew Frank
Mann, Greg
Houssen, Wael
Mykhaylyk, Vitaliy
Duman, Ramona
Thomas, Louise
Jaspars, Marcel
Wagner, Armin
Naismith, James H.
Keywords
Cyanobactins
Azoline oxidase
S-SAD
RIPPs
Phasing
Structure
Sulfur
Long wavelength
QD Chemistry
DAS
Metadata
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Abstract
Determination of protein crystal structures requires that the phases are derived independently of the observed measurement of diffraction intensities. Many techniques have been developed to obtain phases including heavy atom substitution, molecular replacement and substitution during protein expression of the amino acid methionine with selenium methionine. Although the use of selenium containing methionine has transformed experimental determination of phases it is not always possible, either because the variant protein cannot be made or does not crystallise. Phasing of structures by measuring the anomalous diffraction from sulfur atoms, could in theory be almost universal since almost all proteins contain methionine or cysteine. Indeed many structures have been solved by the so called native sulfur single-wavelength anomalous diffraction (S-SAD) phasing method. However, the anomalous effect is weak at the wavelengths where data are normally recorded (between 1 and 2 Å) and this limits the potential of this method to well diffracting crystals. Longer wavelengths increase the strength of the anomalous signal but at the cost of increasing air absorbance and scatter which degrades the precision of the anomalous measurement consequently hindering phase determination. A new instrument, the long-wavelength beamline I23 at Diamond Light Source was designed to work at significantly longer wavelengths compared to standard synchrotron beamlines to open the native S- SAD method for projects of increasing complexity. We report the first novel structure, that of the oxidase domain involved in production of the natural product patellamide, solved on this beamline using data collected at a wavelength of 3.1 Å. The oxidase is an example of a protein that does not crystallise as the selenium variant and no suitable homology model for molecular replacement was available. Initial attempts collecting anomalous diffraction data for native sulfur phasing on a standard macromolecular crystallography beamline using a wavelength of 1.77 Å did not yield large enough signal and S-SAD phasing failed. The new beamline thus has promise in facilitating structure determination by native S-SAD phasing for challenging cases with modestly diffracting crystals and low sulfur content.
Citation
Bent , A F , Mann , G , Houssen , W , Mykhaylyk , V , Duman , R , Thomas , L , Jaspars , M , Wagner , A & Naismith , J H 2016 , ' Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at the Diamond Light Source Beamline I23 by means of S-SAD ' , Acta Crystallographica Section D: Structural Biology , vol. 72 , no. 11 , pp. 1174-1180 . https://doi.org/10.1107/S2059798316015850
Publication
Acta Crystallographica Section D: Structural Biology
Status
Peer reviewed
DOI
https://doi.org/10.1107/S2059798316015850
ISSN
2059-7983
Type
Journal article
Rights
Copyright the Authors 2016. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.
Description
This research was supported by grants from the UK Biotechnology and Biological Research Council (no. BB/K015508/1, J.H.N. and M.J.) and the European Research Council (no. 339367, J.H.N. and M.J.). Mass spectrometry analysis was carried out by the Biomedical Sciences Research Complex Mass Spectrometry and Proteomics Facility, University of St. Andrews and was funded by the Wellcome Trust (grant nos. 094476/Z/10/Z and WT079272AIA). J.H.N. is a Royal Society Wolfson Merit Award Holder and 1000 talent scholar at Sichuan University.
Collections
  • University of St Andrews Research
URI
http://hdl.handle.net/10023/9769

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