Structure determination of the C-terminal fragment of yeast Ski7 using twinned crystal data

Abstract

The structure determination using twinned crystals is challenging although several algorithms have been developed fordetwinning the X-ray data. Our crystal of the C-terminal domain 2 and 3 of Ski7 (Ski7-D2/3), a key part of non-stop mRNAdecay has a perfect twin with the twin operator [h, -h-k, -l]. Many different efforts for phasing with multiple anomalousdispersion techniques using selenomethionine substituted wild-type and mutant proteins were not successful andthe phases were obtained through the molecular replacement method using recently reported structure of C-terminalGTPase domain of Ski7 from Saccharomyces cerevisiae. The overall structure of Ski7-D2/3 is very similar to that of thecorresponding domain of ribosome-associated GTPases including eIF5B, eEF1α, and eRF3. Domains 2 and 3 form aβ-barrel structure containing several structurally deviated long connecting loops. Although the linker between domain 2and 3 is very flexible, the relative orientation between them is virtually the same among all structures, showing that theSki7-D2/3 does not show major conformational movement upon contacting with G domain.