Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriae.
As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His(6) tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5 A resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3 A resolution using one crystal of ShuA-Pb, and at 3.2 A resolution at an energy remote from the Pb M absorption edges for phasing on PROXIMA-1 at SOLEIL.
Brillet
Karl
K
Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, FRE 3211, CNRS-Université de Strasbourg, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
Meksem
Ahmed
A
Thompson
Andrew
A
Cobessi
David
D