On Monday, 14 September 2020 22:22:34 PDT Rezaul Karim wrote:
> Hi Eleanor,Thanks for asking.
> Yes, 2nd domain refine alright with Rfree ~25%, inhibitor at the binding site
> and no major issues at all except that I don't see any trace of 1st domain
> and the protein from dissolved crystals are on SDS gel at right MW size =
> 1st+2nd domain.I tried all other possible space group of P3. Only P31 works
> with ~26% Rfree but with 2 monomers of BD2. Strangely, I find this phenomenon
> only in presence of a specific inhibitor. Unliganded protein structure ~2A
> and with few other inhibitors (1.5-2.2A) all have two domains. Just can't
> wrap my head around the mystery.
Rfree at 0.25 sounds pretty good to me. "Good" as in "good enough to
indicate that you have already described most of the ordered cell content".
Does the lattice packing make sense with only that one domain placed?
I.e., are there enough contacts to make a crystal hold together as a crystal
in the absence an ordered seconds domain?
Ethan
> Thanks,Reza
>
> Md Rezaul Karim
> PhD candidate
> PhD Program in Integrated Biomedical Sciences
> Dept. of Molecular Medicine, Morsani College of Medicine, USF,Tampa
> Schonbrunn lab, Moffitt Cancer Center, Tampa
> Email: rez...@health.usf.edu, md.ka...@moffitt.org
> Phone: (813) 745 4673 ext. 5462
>
>
> On Mon, Sep 14, 2020 at 5:50 AM, Eleanor Dodson<eleanor.dod...@york.ac.uk>
> wrote: Rezaul: You sayBut 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 FellowH. Lee Moffitt Cancer Center and Research
> InstituteTampa, 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 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
>
>
>
>
>
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--
Ethan A Merritt
Biomolecular Structure Center, K-428 Health Sciences Bldg
MS 357742, University of Washington, Seattle 98195-7742
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