REFINEMENT OF THE TWINNED CRYSTAL STRUCTURE OF A PROTEIN-LIKE SMALL MOLECULE WITH 850 ATOMS IN THE ASSYMETRIC UNIT STARTING FROM AN AB INITIO SOLUTION

T.R. Schneider, J. Kärcher, E. Pohl, P. Lubini, and G.M. Sheldrick

Institut für Anorganische Chemie, Universität Göttingen, Tammanstr. 4, 37077 Göttingen, Germany
http://shelx.uni-ac.gwdg.de/~trs, thomas.schneider@shelx.uni-ac.gwdg.de

Keywords: lantibiotics, merohedral twins, ab initio, refinement, restraints

Merohedral twinning is a frequently observed phenomenon in both small molecule and protein crystallography. While small molecule crystallographers consider merohedral twinning more or less as an interesting complication, many protein crystallographers regard it as an unsurmountable problem. However, if a starting phase set can be obtained by e.g. molecular replacement methods, refinement of protein structures against twinned data becomes feasible.

Here we describe the structure solution and refinement of mersacidin, a lantibiotic effective against gram-positive bacteria. A single molecule of mersacidin contains 20 amino acids, about half of which are post-translationally modified. The compound crystallizes in the space-group P3₂ pretending to be P3₂21 as a result of merohedral twinning. The asymmetric unit contains six molecules resulting in a crystallographic problem equivalent to a protein of 120 residues. At cryogenic temperatures crystals diffracted to 1.1 A on BW7B at EMBL Hamburg (100% complete, R_m = 4.9%).

For structure solution, the data were very crudely deconvoluted into contributions arising from the different twin domains. The ab initio program SHELXD[1] succeeded in solving the structure in about four days on a Pentium computer, allowing three quarters of the atoms to be automatically located in the first electron density map.

Starting from this model, refinement was performed against the original twinned data using the program SHELXL[2] employing a mixture of small molecule and protein techniques. Key steps were the introduction of tight restraints on the non-standard amino acids exploiting the local non-crystallographic symmetry ('SADI'-restraints) and substantial phase improvement by automatic placement of dummy atoms in a way similar to ARP[3].

The current crystallographic R-value is 13.6%. The atomic model reveals an extremely tight packing including a non-crystallographic two-fold axis and a set of cleavage planes that provide a possible explanation for the merohedral twinning.

[1] Sheldrick GM, Gould RO: Structure Solution by iterative peaklist optimization and tangent expansion in space group P1, Acta Cryst. B:423-431 (1995).
[2] Sheldrick GM, Schneider TR: SHELXL: High Resolution Refinement, Methods in Enzymology, 277:319-343 (1997).