Dear Rhys,
the humidity control device at Diamond and ESRF (HC1) is apparently
especially recommended for crystals that show large variability. You
might want to give this a try, collect a few wedges at RT off many
crystals and then Blend them. This approach might increase resolution
and, a la W. Hendrickson as mentioned already, signal.
Andreas
On 27/07/2014 10:48, RHYS GRINTER 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
--
Andreas Förster
Crystallization and X-ray Facility Manager
Centre for Structural Biology
Imperial College London
