Solving a 300 kDa multimeric protein by low-resolution MAD phasing and averaging/phase extension

The structure of the conjugative coupling protein TrwB Delta N70 from Esche richia coli plasmid R388 was solved using two crystal forms. This large mul timeric membrane protein of 437 residues per monomer is involved in cell-to -cell single-strand DNA transfer. Diffraction data to 2.4 Angstrom were ava ilable from trigonal crystals obtained from ammonium sulfate and to 2.5 Ang strom from monoclinic crystals grown from tartrate. A single tantalum bromi de (Ta6Br122+) derivative of the trigonal form, which presented a protein h examer with C6 local symmetry in the asymmetric unit, was used in a three-w avelength MAD experiment to achieve 4.5 Angstrom resolution for initial pha ses. Sixfold averaging and phase extension increased the effective phasing resolution and eventually produced a straightforwardly traceable electron-d ensity map. The monoclinic structure was solved by molecular replacement, i .e. a hexamer of the trigonal form was used as a search model. Two such hex amers are present in the asymmetric unit.