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

The structure determination using twinned crystals is challenging although several algorithms have been developed for detwinning 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 mRNA decay has a perfect twin with the twin operator [h, -h-k, -l]. Many different efforts for phasing with multiple anomalous dispersion techniques using selenomethionine substituted wild-type and mutant proteins were not successful and the phases were obtained through the molecular replacement method using recently reported structure of C-terminal GTPase domain of Ski7 from Saccharomyces cerevisiae. The overall structure of Ski7-D2/3 is very similar to that of the corresponding domain of ribosome-associated GTPases including eIF5B, eEF1α, and eRF3. Domains 2 and 3 form a β-barrel structure containing several structurally deviated long connecting loops. Although the linker between domain 2 and 3 is very flexible, the relative orientation between them is virtually the same among all structures, showing that the Ski7-D2/3 does not show major conformational movement upon contacting with G domain. © 2017 Biodesign22Nkciothe

ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils

The coiled-coil (CC) domain is a very important structural unit of proteins that plays critical roles in various biological functions. The major oligomeric state of CCs is a dimer, which can be either parallel or antiparallel. The orientation of each α-helix in a CC domain is critical for the molecular function of CC-containing proteins, but cannot be determined easily by sequence-based prediction. We developed a biochemical method for assessing differences between parallel and antiparallel CC homodimers and named it ACCORD (Assessment tool for homodimeric Coiled-Coil ORientation Decision). To validate this technique, we applied it to 15 different CC proteins with known structures, and the ACCORD results identified these proteins well, especially with long CCs. Furthermore, ACCORD was able to accurately determine the orientation of a CC domain of unknown directionality that was subsequently confirmed by X-ray crystallography and small angle X-ray scattering. Thus, ACCORD can be used as a tool to determine CC directionality to supplement the results of in silico prediction. © The Author(s) 20171101sciescopu