Research Article: Data collection with a tailored X-ray beam size at 2.69 Å wavelength (4.6 keV): sulfur SAD phasing of Cdc23Nterm

Date Published: March 01, 2016

Publisher: International Union of Crystallography

Author(s): Michele Cianci, Matthew R. Groves, David Barford, Thomas R. Schneider.

http://doi.org/10.1107/S2059798315010268

Abstract

Data collection with a tailored 50 µm diameter X-ray beam at 4.6 keV (λ = 2.69 Å) on the newly established EMBL beamline P13 at PETRA III allowed the crystal structure determination of the Cdc23Nterm homodimer (65.4 kDa; 12 Cys and ten Met residues) by sulfur SAD phasing at 3.1 Å resolution while overcoming crystal twinning.

Partial Text

Choosing the wavelength to perform a sulfur SAD (S-SAD) experiment requires thorough consideration. Early landmark S-SAD experiments on crambin (Hendrickson & Teeter, 1981 ▸) and lysozyme (Dauter et al., 1999 ▸) were performed at a wavelength of 1.54 Å using a rotating-anode generator or a synchrotron, respectively, as an X-ray source. During the last decade, the availability of widely tunable beamlines around the world has allowed the use of longer wavelengths or softer X-rays (Djinovic Carugo et al., 2005 ▸; Table 1 ▸). Novel structures such as apo crustacyanin C1 (Gordon et al., 2001 ▸), trypa­redoxin II from Crithidia fasciculata (Micossi et al., 2002 ▸) and an IGF2R fragment (Brown et al., 2002 ▸) have been phased by S-SAD at 1.77 Å wavelength at the ESRF, France. At DORIS III, DESY, Germany, data were collected using an X-ray wavelength of 1.74 Å to solve the structure of the nucleocapsid protein of Porcine reproductive and respiratory syndrome virus (PRRSv; Doan & Dokland, 2003 ▸). At the SRS, Daresbury, England, an S-SAD data set collected at a wavelength of 2.0 Å, in combination with a xenon-derivative set, was used to provide phase information for apo crustacyanin A1 (Cianci et al., 2001 ▸). At XRD1 Elettra, Italy, the structure of DsvC was solved by collecting data at a wavelength of 1.9 Å (Weiss et al., 2004 ▸). On the X12C beamline at the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, New York, USA), the structure of an FMN reductase from Pseudomonas aeruginosa PA01 was determined using an S-SAD data set collected at a wavelength of 1.7 Å (Agarwal et al., 2006 ▸). The structure of VEGF-E was determined by S-SAD using a data set collected at a wavelength of 1.7 Å at the SLS, Villigen, Switzerland (Wagner et al., 2006 ▸). On the PROXIMA 1 beamline at the SOLEIL synchrotron, Saint Aubin, France, de novo chlorine/sulfur SAD phasing of a structural protein from ATV was achieved by collecting data at a wavelength of 2 Å (Goulet et al., 2010 ▸).

The provision of a 2.69 Å wavelength (4.6 keV) beam with variable beam size was instrumental for the structure solution of Cdc23Nterm at 3.1 Å resolution via the anomalous signal from S atoms intrinsic to the protein. A variable micrometre-size beam allowed the collection of sulfur data from separate regions of a crystal, which would otherwise be discarded, thus overcoming crystal imperfections owing to the presence of two differently oriented twin domains. The long wavelength enhanced the Bijvoet ratio to 2.21% (1.0% at 7 keV), thus increasing the anomalous signal to a level where it was possible to determine the sulfur substructure accurately even with moderate data multiplicity. The quality of the calculated phases allowed complete model autotracing at a resolution as low as 3.1 Å.

 

Source:

http://doi.org/10.1107/S2059798315010268

 

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