Dear Pravin, this brings me to re-emphasize a point I've just made at RapiData. If you're working with PILATUS or EIGER detectors, there is no good reason not to spread your dose over at least 360° of data. You will get better statistics than if you collect 57.2° or whatever your strategy software recommends - and you can laugh P1 in the face if it comes your way. Just make sure to adjust expose time or attenuation accordingly when expanding your data collection to 360°.
This is of no help after data collection, but since you collected data for SeMet SAD, you probably already have 360° or more and can thus work in P1 (as James suggested) without much pain. All best. Andreas On Thu, Apr 20, 2017 at 12:11 AM, James Holton <jmhol...@slac.stanford.edu> wrote: > > In situations like this I always try dropping to P1. Even if the data are > highly incomplete in P1 you can still refine it. Difference maps are > degraded by poor completeness, but you still might see something. But > either way, the R-factors will tell you something. if dropping to P1 > solves your R-factor problems then you know you were in the wrong space > group. > > The biggest caveat to dropping symmetry operators (and essentially > replacing them with NCS operators) is that you want to be VERY careful not > to mix your working and free sets. The best way to do this is pick your > free set with the highest-possible symmetry given your lattice, and then > expand that to P1 using the CCP4 program "CAD". Then you change the space > group in "CAD" and it will chunk out the right asymmetric unit. Oh, and > then use PDBSET to expand your model to P1 as well. > All this is done automatically by the program Zanuda, but even Zanuda > cannot un-merge data. You need to provide the P1 structure factors and > then see what it tells you. > > Does that help? > > -James Holton > MAD Scientist > > On 4/18/2017 5:56 PM, gnufreebsd wrote: > > Dear Pravin > for a kinase, n lobe is quite flexible , especially beta1 and beta2, and > residues beyond theses two strands. > > > best regards > tiger > > 发自 WPS邮箱客戶端 <http://mo.wps.cn/wps-mail/mail.html> > 在 Pravinkumar Jagtap <pravinja...@gmail.com> <pravinja...@gmail.com> > ，2017年4月12日 上午2:55写道： > > Dear All, > I am stuck with this problem for 2 months and hope you could help. > > We have a 2.1 A dataset for a 380 amino acid long protein. The space group > is I4 (single molecule in asymmetric unit, 48% solvent content) and the > dataset is quite perfect (no obvious pathologies). The protein itself is > organised in 2 lobes (N and C terminal lobes). The sequence identity to > nearest homologue structure is 17%. > > We could get the phases by SeMet SAD phasing (3A resolution dataset, 5 > SeMet (excluding N-terminal Met), 3 full occupancy SeMet in C-terminal lobe > and 2 partial occupancy (~0.5 each; present on surface) SeMet in N-terminal > lobe). Automated model building (at 2.1 A) yielded nice model for the > C-terminal lobe (215 residues) and manually I could build parts (around 80 > residues) of N-terminal lobe with high confidence. In addition we could > also build a ligand which is sandwiched between C and N terminal lobe. > > However the Rfree is stuck at 0.39 (Rwork 0.33). There is indeed some > patchy density left at the N terminal lobe but as it is discontinuous, I > cannot build anything in it (except lots of water molecules). In total I am > missing around 85 residues. These residues are predicted to be present in > secondary structure (and not flexible). > > As I have around 75-80% model built, I would expect that I would have all > the phases and should get nice density for the remaining part. But as I > dont see it, could the rest part be flexible? But again, this is not > reflected in the R factors (I would then expect low Rfree). > > Could it be that I still lack phases (due to partial occupancy of SeMeth > in N-terminal lobe ) and have to try to get them by heavy metal soaking, or > there is disorder in the N-terminal lobe? I have also tried solving > different datasets for same crystal but this has not been useful. > > Regards, > Pravin. > > > -- <https://www.dectris.com> Andreas Förster, Ph.D. MX Application Scientist, Scientific Sales Phone: +41 56 500 2100 <+41%2056%20500%2021%2000> | Direct: +41 56 500 21 76 <+41%2056%20500%2021%2076> | Email: andreas.foers...@dectris.com DECTRIS Ltd. | Taefernweg 1 | 5405 Baden-Daettwil | Switzerland | www.dectris.com [image: LinkedIn] <https://www.linkedin.com/company/5067919> [image: facebook] <https://www.facebook.com/pages/Dectris-Ltd/623855944369304> <https://twitter.com/DECTRIS_News> Confidentiality Note: This message is intended only for the use of the named recipient(s) and may contain confidential and/or privileged information. If you are not the intended recipient, please contact the sender and delete the message. Any unauthorized use of the information contained in this message is prohibited.
