Dear William,
In a similar circumstance, we cut out the density from one crystal, and used that to solve the structures of non-isomorphous crystals by molecular replacement. At this point, we had an envelope (needed to cut out the density), initial maps, and operators to place one copy of the density in the other crystals, which is all you need for multi-crystal averaging. The more non-isomorphous your crystals are, the more powerful this is. What’s important (Kevin Cowtan explained this in a talk at the IUCr meeting in Glasgow) is that the size of the difference in cell dimensions should preferably be comparable to the resolution of the data (dmin) or larger. Our case was a bit difficult because the non-isomorphism was marginal — enough to stop us from using MIR(AS) but barely enough to help the phasing.
There are other cases of people using similar approaches in the literature.
Best wishes,
Randy Read
-----
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
Wellcome Trust/MRC Building Fax: +44 1223 336827
Hills Road E-mail: [log in to unmask]
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
On 18 Feb 2015, at 12:51, William Chao <[log in to unmask]> 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.
|