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Experimental phasing with vanadium and application to nucleotide-binding membrane proteins

DOI: 10.1107/S2052252520012312 DOI Help

Authors: Kamel El Omari (Diamond Light Source; Research Complex at Harwell) , Nada Mohamad (University of Oxford) , Kiran Bountra (Research Complex at Harwell; Imperial College) , Ramona Duman (Diamond Light Source; Research Complex at Harwell) , Maria Romano (Imperial College London; Research Complex at Harwell; National Research Council (IBB–CNR)) , Katja Schlegel (University of Oxford) , Hok-sau Kwong (Research Complex at Harwell; Imperial College) , Vitaliy Mykhaylyk (Diamond Light Source; Research Complex at Harwell) , Claus Olesen (Aarhus University) , Jesper Vuust Moller (Aarhus University) , Maike Bublitz (University of Oxford) , Konstantinos Beis (Research Complex at Harwell; Imperial College) , Armin Wagner (Diamond Light Source; Research Complex at Harwell)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Iucrj , VOL 7

State: Published (Approved)
Published: November 2020

Open Access Open Access

Abstract: The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can be difficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein–vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein–vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.

Journal Keywords: experimental phasing; vanadium; membrane proteins

Subject Areas: Biology and Bio-materials, Technique Development

Instruments: I23-Long wavelength MX