Re: [ccp4bb] crystals of 1D
In case you are using a standard purification protocol (with, say, two purification steps and a polishing gel filtration) you might also try adding yet another purification step, no matter how pure your sample looks on SDS-PAGE. Tim -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] molprobity in coot: BL WARNING:: reduce didnt run ok, so stop here!
Hello Rongjin Guan, your problem most likely comes from different pdb version formats. The files which work are probably pre pdb v. 3.0. Although (Win)Coot is mainly v. 3 compliant here we are not (really (*)). A fix (at least for the files which are not working) may be to change two lines (198/199) in the file 'generic_objects.py' (in YOURWINCOOTDIRECTORY\share\coot\python) Change from: 198: # [-build, mol_pdb_file], 199: [-build, -oldpdb, mol_pdb_file], to 198: [-build, mol_pdb_file], 199: # [-build, -oldpdb, mol_pdb_file], Hope this helps, B (*) note to self (and Paul) we probably should have this as an optional parameter and/or ideally automatically detected which pdb version we are using and adjust the probe parameters accordingly?! Rongjin Guan wrote: Hello, I just installed Wincoot 0.6.1 and reduce/probe as well, and tested with several PDB files for probe/clash validation. For some PDBs it worked perfectly; but for my own model, it did not work and I have the following message: . Found 0 hydrogens (0 hets) Standardized 0 hydrogens (0 hets) Added 3946 hydrogens (0 hets) Removed 0 hydrogens (0 hets) Adjusted 113 group(s) If you publish work which uses reduce, please cite: Word, et. al. (1999) J. Mol. Biol. 285, 1735-1747. For more information see http://kinemage.biochem.duke.edu BL WARNING:: reduce didnt run ok, so stop here! run_generic_script (probe, 0) My model was outputed from phenix refinement and I checked the format and can not see anything wrong. Can anyone give me some hints? Thanks Rongjin Guan
[ccp4bb] Phasing statistics
Dear CCP4ers, I've been asked by a referee to provide the phasing statistics for a SAD dataset that I used to solve a recent structure. Whilst I have been able to find a figure-of-merit for the data after phasing, I can't work out how to get any other statistics (e.g. phasing power or an equivalent or Rcullis). Does anyone know a good route to obtaining useful statistics to put in the paper for SAD data? The structure solution was carried out using SHELX C/D/E and then ARP/wARP. Thanks in advance, Nic Harmer = Dr. Nic Harmer School of Biosciences University of Exeter tel: +44 1392 725179
[ccp4bb] protein degradation during concentration for crystallization trials
Dear all I am working on purification of 14 kd protein(pI 8.3, basic protein) that has MBP(maltose binding protein, 45 kd,) tag, and same protein in other vector(pGEX-KT) that has GST tag. During affinity purification in both cases I used 300mM nacl and 50 mM tris, pH 7.5 buffer throughout the purification.I do on column cleavage with TEV to remove the tag (for crystallization purpose). Purification with MBP tag: After cleavage MBP also appear in elution fraction along with cleaved protein of my interest. To remove MBP(pI 5.0) from protein of my interest I have to do anion exchange chr. with DEAE resin(weak anion exchanger) with buffer of pH7.3. Hence I do dialysis against the buffer 20mM nacl and 20 mM tris, ph 7.3. in cold room. I have few problems: 1) why the MBP elute with protein of my interest ( I tried at low salt concentration also but still elute). 2) During dialysis my protein of interest precipitate. 3) If I tried FPLC, protein of interest and MBP elute in same fraction with superdex 200 column. Problem with GST tag purification: it give me impure protein even after enough washes with high salt concentration buffer (upto 2 molar). When i concentrate protein in CENTRICON (Millipore, centrifugation 4000rpm at 4 degree, 300mM NaCl and 50 mm Tris buffer with 5mM BME) it degrade very fast and probably aggregate as smear obtain on SDS page below the size of protein after concentration (even protease inhibitor not very much helpful) and band intensity of protein of interest almost remain same before and after concentration step. Please send me your valuable suggestion to overcome to these difficulty. I have also tried with some additives such as sucrose, glycerol, PBS buffer. With regards -- $$ VIKRANT Junior Research fellow Cancer Research Institute Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai, India $$$ Send free SMS to your Friends on Mobile from your Yahoo! Messenger. Download Now! http://messenger.yahoo.com/download.php
Re: [ccp4bb] protein degradation during concentration for crystallization trials
Hi, MBP tag: 1.there might be a TEV cleavage site in your MBP variant. 2. your protein needs salt to stay in solution 3. your protein forms aggregates with MBP GST tag: you probably concentrate a protease together with your protein. You need a protease inhibitor kit to take care of different types of proteases. It looks like a His tag would be a better option for you. Maia vikrant saa wrote: Dear all I am working on purification of 14 kd protein(pI 8.3, basic protein) that has MBP(maltose binding protein, 45 kd,) tag, and same protein in other vector(pGEX-KT) that has GST tag. During affinity purification in both cases I used 300mM nacl and 50 mM tris, pH 7.5 buffer throughout the purification.I do on column cleavage with TEV to remove the tag (for crystallization purpose). Purification with MBP tag: After cleavage MBP also appear in elution fraction along with cleaved protein of my interest. To remove MBP(pI 5.0) from protein of my interest I have to do anion exchange chr. with DEAE resin(weak anion exchanger) with buffer of pH7.3. Hence I do dialysis against the buffer 20mM nacl and 20 mM tris, ph 7.3. in cold room. I have few problems: 1) why the MBP elute with protein of my interest ( I tried at low salt concentration also but still elute). 2) During dialysis my protein of interest precipitate. 3) If I tried FPLC, protein of interest and MBP elute in same fraction with superdex 200 column. Problem with GST tag purification: it give me impure protein even after enough washes with high salt concentration buffer (upto 2 molar). When i concentrate protein in CENTRICON (Millipore, centrifugation 4000rpm at 4 degree, 300mM NaCl and 50 mm Tris buffer with 5mM BME) it degrade very fast and probably aggregate as smear obtain on SDS page below the size of protein after concentration (even protease inhibitor not very much helpful) and band intensity of protein of interest almost remain same before and after concentration step. Please send me your valuable suggestion to overcome to these difficulty. I have also tried with some additives such as sucrose, glycerol, PBS buffer. With regards -- $$ VIKRANT Junior Research fellow Cancer Research Institute Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai, India $$$ ** ** Send free SMS to your Friends on Mobile from your Yahoo! Messenger. Download Now! http://messenger.yahoo.com/download.php
[ccp4bb] Follow up to TLS, NCS and refinement
Hello again! Following my previous question, there was something wrong with the staring model for molecular replacement. Now that is sorted, I have 8 complexes in the ASU. After a few rounds of refinement with NCS and isotropic Bfactors, both the Rfactor and Rfree get stuck at 30% and 36%, respectively. I have only just noticed that I am trying to model ~43000 atoms with ~95000 unique reflections (98% complete, at 2.58 Angstrom resolution). Is this the reason why the R factors are stuck and I should start the refinement again from scratch using overall Bfactors with NCS and switch to TLS once my Rfactor is less than 30% and possibly reduce the number of reflections used for Rfree (currently using the standard CCP4 5%) Any input would be greatly appreciated! Dan
Re: [ccp4bb] Follow up to TLS, NCS and refinement
On Thu, Apr 8, 2010 at 3:26 PM, Daniel Bonsor bon...@bbri.org wrote: Following my previous question, there was something wrong with the staring model for molecular replacement. Now that is sorted, I have 8 complexes in the ASU. After a few rounds of refinement with NCS and isotropic Bfactors, both the Rfactor and Rfree get stuck at 30% and 36%, respectively. I have only just noticed that I am trying to model ~43000 atoms with ~95000 unique reflections (98% complete, at 2.58 Angstrom resolution). Is this the reason why the R factors are stuck and I should start the refinement again from scratch using overall Bfactors with NCS and switch to TLS once my Rfactor is less than 30% and possibly reduce the number of reflections used for Rfree (currently using the standard CCP4 5%) I don't think it's a parameters-to-data-ratio problem - the combination of NCS, geometry, and ADP restraints will help prevent overfitting, and the difference between R and R-free isn't unreasonable (they're just both too high). At 2.58A isotropic B-factors are very appropriate, and switching to overall (per-residue?) will probably make everything worse. There is a long list of other things to check first, starting with twinning and/or other undiagnosed crystal pathologies such as pseudosymmetry. I'm not sure what to suggest about TLS refinement, but now would be a good time to try it and see if it helps. You could also try adjusting the strength of NCS restraints, but I doubt this will make a huge difference either way. Using a smaller test set will almost certainly not help anything. I've heard arguments that you can get away with less than 5% of reflections in the test set, if the resulting number is at least 2000, but personally, I'd keep 5%, if only to avoid the inevitable sniping by reviewers. -Nat
