Re: [ccp4bb] Cys modification - deciding between CSS and SMC
Hi Mohamed, Did you try CSO or CSD? I've seen oxidation of Cys residues in the active site of Cysteine-dependent Phosphatases before. In addition, oxidation is more easily explainable than CSS and SMC, especially if reducing agents were omitted from final purification steps. HTH, Dave -- Dr David C Briggs Hohenester Lab Department of Life Sciences Imperial College London UK http://about.me/david_briggs From: Mohamed Noor Sent: Sunday, 21 May, 13:00 Subject: [ccp4bb] Cys modification - deciding between CSS and SMC To: ccp4bb@jiscmail.ac.uk I am working on a model at a resolution of 2.1 A. The active site Cys in both copies have a positive density towards the S end of the residue and these blobs are there in FEM and Polder maps. When I replace these residues with either CSS or SMC, I get the following statistics. What is the best way of deciding which one is correct? CSS: R/Rfree - 0.2016/0.2299 Local CC (chain A/B) - 0.950/0.937 Bfactor (chain A N/CA/C/O/CB/SG/SD) - 42.8/48.6/46.7/42.8/57.3/66.2/98.8 Bfactor (chain B N/CA/C/O/CB/SG/SD) -42.0/44.5/47.2/45.7/57/60.3/95.3 No residual positive density left. SMC: R/Rfree - 0.2087/0.2299 Local CC (chain A/B) - 0.903/0.886 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 43.9/47.9/46.5/42.1/51.8/64.4/66.5 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 43.4/45.0/47.3/45.4/51.8/59.4/66.1 Crescent shaped positive density left towards the CS atom end. Although CSS seems more plausible, the high B factors of SD make me wonder if they are correct. Thanks. Mohamed
[ccp4bb]
Thanks for the suggestion about using NCS constraint to apply change on one subunit to other subunits. That should make life much easier. About the problem of phenix realspace refinement, I figured it out. I used GUI and when I checked the log file, the "ramachandran_restraints" was turned off. It is odd since there does not seem to have any buttons to turn "ramachandran_restraints" off in GUI. Then I tried command line, the Ramachandron statistics look normal now ("ramachandran_restraints" is on in log file). Thanks. On Fri, May 19, 2017 at 11:01 AM, Pavel Afoninewrote: > > > Yes, that is what I have been doing. Build one subunit and assemble into >> tetramer before realspace refinement (with "ncs" constraints). I used >> tetramer for refinement because I want the distances between the inter >> subunits interaction partners to be considered. The problem is, whenever I >> want to make some manual adjustments of the model afterwards, I have to >> break it into monomer and repeat the whole process again. I assume if the >> map is in P4 spacegroup, I only need to modify one subunit of this protein >> and changes will be automatically applied to the other subunits. Am I >> right? >> > > So you have 4 copies and NCS group consisting of one master (or reference) > copy and three related by NCS symmetry. Then you make changes in master > copy and when you run refinement with NCS constraints the changes will be > propagated onto the related copies by applying NCS constraints. This is how > this works in phenix.real_space_refine. So no need to make identical > changes in all 4 copies or go to P4. > > > >> Another question is, when I tried to refine my model using phenix >> realspace refinement (energy minimization and adp), the statistics >> generally become better (map CC, rotamer outlier etc), however, the >> ramachandron statistics become worse, with increase number of outliers >> (from below 1% to around 5%) and allowed (from several percent to 10-20%). >> Do you know what could be the cause? I know I am asking the right person... >> > > This is not expected (about worsening Ramachandran plot outliers). I'm > sure we can solve this problem if you send me model and map files (and > resolution) off list (if files too large to send by email please use > Dropbox or similar file sharing tools). > > Pavel > >
Re: [ccp4bb] crystallization screen for protein-protein complex
Dear Hena, You already have excellent advice but I would also look at the PEG smear approach developed by Frank von Delft’s group – Chaikuad et al., Acta Cryst D. 7, 1627-39 (2015). It’s commercially marketed by Molecular Dimensions and has features which may be amenable to complex systems. Caveat emptor, we don’t have any data on any of the academic samples that have been submitted to our crystallization center to vouch for its success but we would suggest taking a look. If you are interested in testing a lot of conditions in an efficient way the high-throughput crystallization screening center at our institute uses a lot of the commercially available screens (http://hwi.buffalo.edu/crystallization-cocktails/) and covers a fairly extensive area of chemical space. That can save a lot of time and effort. Details are at http://getacrystal.org. Good luck, Eddie Edward Snell Ph.D. President and CEO Hauptman-Woodward Medical Research Institute Assistant Prof. Department of Structural Biology, University at Buffalo 700 Ellicott Street, Buffalo, NY 14203-1102 hwi.buffalo.edu Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu [cid:image003.png@01D2D241.8F507E50] Heisenberg was probably here! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hena Dutta Sent: Saturday, May 20, 2017 5:15 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization screen for protein-protein complex Dear Members, I am trying to crystallize a protein-protein complex. Both are soluble proteins and form complex as observed in FPLC either co-purifying or separately purifying and then mixing with equi-molar ratio. Looking for suggested screens to try for crystallization. So far tried with Qiagen - ProComplex Suite, Classics Suite and Classics II Suite No success yet. Is there any guidance which screen to try? Looking for your help... Regards, Hena.
[ccp4bb] Cys modification - deciding between CSS and SMC
I am working on a model at a resolution of 2.1 A. The active site Cys in both copies have a positive density towards the S end of the residue and these blobs are there in FEM and Polder maps. When I replace these residues with either CSS or SMC, I get the following statistics. What is the best way of deciding which one is correct? CSS: R/Rfree - 0.2016/0.2299 Local CC (chain A/B) - 0.950/0.937 Bfactor (chain A N/CA/C/O/CB/SG/SD) - 42.8/48.6/46.7/42.8/57.3/66.2/98.8 Bfactor (chain B N/CA/C/O/CB/SG/SD) -42.0/44.5/47.2/45.7/57/60.3/95.3 No residual positive density left. SMC: R/Rfree - 0.2087/0.2299 Local CC (chain A/B) - 0.903/0.886 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 43.9/47.9/46.5/42.1/51.8/64.4/66.5 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 43.4/45.0/47.3/45.4/51.8/59.4/66.1 Crescent shaped positive density left towards the CS atom end. Although CSS seems more plausible, the high B factors of SD make me wonder if they are correct. Thanks. Mohamed
Re: [ccp4bb] crystallization screen for protein-protein complex
Hena There was a very interesting paper by Peter Sun and coworker from 2002. They pointed out that there is a very strong bias towards crystallizing protein-protein complexes with PEG rather than salt as the main precipitant. Patrick ___ Radaev and Sun. Crystallization of protein-protein complexes. J. Appl. Cryst. (2002). 35, 674-676 On 20 May 2017 at 22:14, Hena Duttawrote: > > Dear Members, > I am trying to crystallize a protein-protein complex. Both are soluble proteins and form complex as observed in FPLC either co-purifying or separately purifying and then mixing with equi-molar ratio. > Looking for suggested screens to try for crystallization. > > So far tried with Qiagen - ProComplex Suite, Classics Suite and Classics II Suite > No success yet. > > Is there any guidance which screen to try? > > Looking for your help... > Regards, > Hena. -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36