Hi Rhys, Have you tried quick back-soaks into solutions lacking the heavy atoms? This can reduce radiation decay caused by absorption of X-rays by overabundant heavy atoms.
Best, Chris > On Jul 27, 2014, at 4:48 PM, RHYS GRINTER <r.grinte...@research.gla.ac.uk> > wrote: > > Hi All, > > I thought I might put a question to the community, with the hope of getting > some tips of the best way to proceed with my heavy atom phasing problem. > I'm working on solving the structure of an integral beta-barrel membrane > protein of approximately 100 kDa. I've crystallised protein, growing some > very flimsy needle like crystals, and collected datasets to around 3.1 A. > I then produced selenomet derivative protein and repeated crystallisation > trials in the same conditions and also repeated broad screens, however the > derivative protein failed to produce crystals that diffracted beyond 10 A (in > fact it barely crystallises at all). > So I've moved on to heavy atom soaks and have had some success with > tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals > didn't dissolve (as they did with gold and samarium compounds) and diffracted > to some degree. I collected SAD data to around 6.5 A from these crystals and > there seems to be anomolous signal. However, while I get a good CC of 0.4 > from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps > before and after DM are uninterpretable. I'm guessing the quality and > resolution of the data I collected just aren't good enough (the data is > reasonably anisotropic). > I performed the metal soaking, by taking a small amount of the platinate salt > and adding it to the crystallisation drop as the crystals are extremely > fragile and don't stand up well to handling through a soaking or cryo > solution. Leaving the crystals to soak for 48 hours and then, freezing them > directly. The solution is on the border of cryoprotection (the conditions has > PEG2000MME and PVP and the precipitants), but with native crystals this > doesn't seem to be a parameter which affects diffraction. The crystals are > very variable in performance, so while I feel that the heavy atom soaking has > compromised their diffractability to a degree, inherent variation may play a > part. > > What I was wondering is if some one with more experience than me found > themselves in this position, how would they proceed? Questions which spring > to mind are, how much heavy atom compound do people add and how long do they > soak for? Is there anyway I can squeeze something out of the anomalous data I > have, given I have 'reasonable' native data, or will poor quality data give > spuriously positive statistics for heavy atom phasing? And are there any > tricks people have experienced to improve performance of crystals like these > (aside from the usual seeding, additives, different detergents etc which I > have spend a fair bit of time on optimization already). > > Thanks in advance, > > Rhys
