On the use of the loopless free crystal mounting method for the longer X-ray S-SAD phasing
Faculty of Advanced Life Science,
Hokkaido University, Japan
Single wavelength anomalous diffraction (SAD) phasing has become a potential tool for protein structure determination. The choice of X-ray wavelength is one of the major decisions required for SAD data collection. In the laboratory, however, we may only choose from several anticathode targets. Considering the phasing using the anomalous signal of sulfur atoms of underivatized native protein, longer wavelength of Cr Kα X-rays (2.29 Å) might be an optimal choice, where the anomalous intensity differences or the Bijvoet ratio becomes about 1% of total reflection intensity, because the Δf" value of sulfur becomes 1.14 e- as compared to 0.56 e- at Cu Kα. But the Bijvoet difference is still very small and highly accurate data collection is essential. One of the experimental difficulties in using longer wavelengths is the increased absorption. Especially, in standard protein crystallography where the crystal is mounted in a cryoloop with cryo-buffer, X-ray absorption by these materials sometimes prevents the detection of tiny anomalous signals. We have developed a novel loopless free crystal mounting technique to eliminate absorption by the cryo-buffer and cryoloop. This technique increases the precision of the anomalous differences between the Bijvoet mates, and makes the in-house S-SAD method with a Cr Kα X-ray source a very useful tool for high-throughput structure determination. The practical applicability of in-house structure determination using Cr Kα X-rays and the loopless free crystal mounting method was examined using several novel proteins. Proteins from 9.6 kDa to 84 kDa have been solved using this method without any derivatization. In most cases, more than 90% of all the structures were constructed automatically with side chains by use of the SAD phasing method.