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
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