Hi, If your Hg dataset is not isomorphous with the SeMet you can cut out some density and use cross-crystal averaging (molecular replacement using electron density) to transfer the Hg phases to the Se dataset. You can then try using an anomalous difference fourier map to find more or weaker Se sites. If that manages to get density good enough to even partially build a model, you can put it through multiple rounds of MR-SAD with phaser. Basically you use your model phases to help find more weak Se sites, which improves your phases and allows you to build a better model, and you go around again.
Jason. — Dr Jason Busby Laboratory of Structural Biology School of Biological Sciences The University of Auckland Private Bag 92019 Auckland Mail Centre Auckland New Zealand ph: +64 9 3737599 ext 88958 On 19/02/2015, at 1:51 am, William Chao <william.c...@cancer.org.uk<mailto:william.c...@cancer.org.uk>> wrote: Dear all, I am trying to phase a largish novel structure of 130 kDa with P21 (sometimes P222) space group. So far I have collected a few 3.5ish-Å 3-wavelength MAD datasets from SeMet derivatives as well as an anisomorphous 6Å Hg peak dataset (with detectable anomalous signal). As my crystal is rather anisotropic, CC1/2 of one direction of the Se data drops below 0.5 at 4Å. I can generate a map from a MAD dataset that gives a overall shape of the molecule with clear molecular boundary after solvent flattening, resembling the shape of some EM class averages that I obtained earlier. However, the density of this map is very discontinuous and is impossible to build any helix in by machine or by eye. As I use an insect-cell expression system and the occupancy of Se is expected to be low, the programmes that I used could only find a 2 reasonable sites out of 24 Se per molecule. I have about 10 Se crystals and 50 native crystals (which I shall use for derivatisation) left for one last trip before the synchrotron shuts down for two months. Could someone advise me on a reasonable data collection strategy that could maximise my chance on this upcoming trip? I am sure that many people have encountered difficult data like this one and have solved their structures successfully. Would anyone be able to advise me on how it'd be best to improve my phases/density given the limitations of the data? Also to mention that the best native crystal can diffract to 3A with CC1/2 of the worst direction dropping to 0.5 at 3.5A. Many thanks in advance! William --- NOTICE AND DISCLAIMER This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. Cancer Research UK Registered charity in England and Wales (1089464), Scotland (SC041666) and the Isle of Man (1103) A company limited by guarantee. Registered company in England and Wales (4325234) and the Isle of Man (5713F). Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
