Rezaul: You say *But I only see the 2nd domain with exact same unit cell that I have solved for 2nd domain's with same space group. *
I am afraid if that is so that you probably have only crystallised the 2nd domain. Does that domain refine all right? Eleanor On Mon, 14 Sep 2020 at 09:02, Rezaul Karim < 00001e364c8f16de-dmarc-requ...@jiscmail.ac.uk> wrote: > Eagerly following > > I have a similar case. A 3A data with P3(1)21 from two domains (80+% > sequence identity) protein. No major twinning issue. But I only see the 2nd > domain with exact same unit cell that I have solved for 2nd domain's with > same space group. > No proteolysis- crystals give protein band on SDS at right MW. Data from > 20+ crystals from various condition & additive screens came out all the > same. > Resorted to changing the construct to make variable N/C-term for future. > > Thanks, > Reza > > Md Rezaul Karim > Postdoctoral Research Fellow > H. Lee Moffitt Cancer Center and Research Institute > Tampa, FL > Email: rez...@health.usf.edu, reza.ka...@moffitt.org > Phone: (813) 745 4673 ext. 5462 > > On Sun, Sep 13, 2020 at 7:33 PM, Jon Cooper > <0000488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote: > Hello, a couple of thoughts. > > If your best dataset has a twinning fraction of 40% (i.e. almost 50:50 > ;-?) and the twin operator corresponds to a 2-fold rotation parallel to the > z-axis, is it definitely trigonal, rather than hexagonal? The 2-fold NCS > operator that you found for the d3 domains, is it parallel to an axis, e.g. > z? Also, how long did the crystals take to grow and do you have the > original diffraction images? If so, and I know its hard to tell with > fine-slicing on modern detectors, do they look clean or do they show any > splitting of the spots, etc? Also, a bit boring question, but when you say > the space group is P32, I guess you mean P3 subscript(2) rather than P321, > and I take it you have tried alternative space groups in the MR like P3 > subscript(1). Are the systematic absences good, or is it all a bit > ambiguous? > > Best wishes, Jon Cooper. > jon.b.coo...@protonmail.com > > Sent with ProtonMail <https://protonmail.com> Secure Email. > > ‐‐‐‐‐‐‐ Original Message ‐‐‐‐‐‐‐ > On Tuesday, 8 September 2020 19:53, Andrew Lovering <a.lover...@bham.ac.uk> > wrote: > > Dear all, > > > I've a project with two historical datasets (i.e. not sure of when/if I > can reproduce to solve problem by getting better data) that are twinned. > > > The spacegroup is P32, with operator -h, -k, l > > cell = 83.5 83.5 64.2 90 90 120 > > > protein has three domains d1-d2-d3, likely 50% solvent, 2 copies in asu > > > dataset 1, roughly 3.6 angstrom resolution, twin fraction 0.25 > > dataset 2, roughly 3.1 angstrom resolution, twin fraction 0.4 > > > I've managed to solve this (unambiguously) by finding a d3:d3 dimer in > detwinned data, then seeing via phenix autobuild that it finds a d2 also in > map. At present the ability to interpret the map further stalls there, and > it can't be "the limit" because lack of crystal contacts suggest there > should be more protein to find..... > > > I realise I can go two ways, either using the detwinned data or using > twinned data and supplying the operator.....the former giving ridiculously > optimistic R-values with a suspiciously "clean map", the latter giving > noisier but maybe more informative maps. > > > Two-fold averaging is not going to be great because the d2-d3 arrangement > observed in monomer 1 clashes when placed over monomer 2 (so at present can > only average the small % of ASU between d3 copies). Critically, looking for > a second d2 using MR fails but the lack of crystal contacts suggests > success should be possible....its really unlikely that a third entity is in > there (d1 and d2 are actually 50% similar, so by searching for d2 and > failing, I'm actually missing three spots it could potentially occupy if > the protein is not proteolysed) > > > Sorry for long email but the devil is in the details. My question would > be, what is best practice in a low res poor twinned dataset where you can > only fill roughly just over half of what should be in there? > > > Feel free to tell me its doomed! > > > Best wishes & thanks in advance > > Andy > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
