[ccp4bb] Negative electron Density for Zinc
Hi everybody, I am working on a mutant protein structure which is 56 aminoacids long and solved the structure using SAD( Single wavelength anomalous dispersion) with zinc ions. We used SHELX software to locate the Zinc atoms and solved phases. We got the structure as a monomer. The resolution of the structure is 1.4 A0 . The problem with the structure is, when i calculate the structure factors in COOT it always shows negative electron density around ZINC atoms which have been located by SHELX. I initially thought it was because my COOT software was very old and it doesn't recognize the Zinc atoms( I am new to crystallography) . I calculated the structure factors using FFT from CCP4 and it shows the same. I don't understand the problem. It refines well in Refmac and B-factors also look very good. Anyone know what the problem is? Thank You in advance Deepthi -- Deepthi
[ccp4bb] Negative electron Density for Zinc
Hi Everybody Thank You very much for your suggestions. I did play around with Zinc occupancy by giving 0.5 to each of them. I got rid of the negative electron density. But i think it still needs to be refined. Thank You very much once again Deepthi
Re: [ccp4bb] Regarding refinement in Refmac5
Hi Yes i did check my MTZ file header. It shows p3221. For the same reason i reprocessed the data again in both p3221, p3121 and p321. Except for p3221 none of the other space groups fit the model. The packing looks good and the map shows side chains very clearly. When i add the respective side chain its just not accepting. May be it is not the correct solution? Is it possible? Thank You Deepthi On Thu, Jul 19, 2012 at 7:04 AM, Eleanor Dodson eleanor.dod...@york.ac.ukwrote: It isn't that your space group is wrong, but are you sure that your mtz file has that space group in its header? MR will test all possible alternatives - in this case P3121 or P3221 - but won't change the symmetry information in the input mtz. You need to do that with a utility like mtzutils hklin1 p3121.mtz hklout p3221.mtz SYMM P3221 end And there are various options in the REFLECTION UTILITIES. The integration and data processing are exactly the same for either space group, but REFMAC does not check that your mtz and pdb have the SAME symmetry information, and by default uses that in the mtz file. So you could have a perfectly good solution in P3221 but be running refinement in P3121, which is NOT GOOD! Eleanor. On 18 Jul 2012, at 17:50, Deepthi wrote: I tried opening the model with other spacegroups MTZ file. The map doesn't fit well for other spacegroups. The initial model was refined using Phenix Autobuild software. I tried MR with every spacegroup possible in primitive hexagonal. Only p3221 worked. There is no twinning in the crystal. I will try using other softwares for refinement but this is annoying. I also tried mutating the model to poly alanines and refine but this made it worse. The R-free went up to 0.546. I initially thought it might be a space group problem but trying other space groups doesn't work either. Thank youvery much for the help Deepthi On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi there, Not much information provided. How was the initial model refined ? Phenix ? It could be a problem with the Refmac refinement protocol (difficult to say with so little information) if you switched from Phenix to Refmac. How certain are you 1 - of the space group; 2 - that the crystal wasn't twinned ? You can have both and it can be annoying. Further, at this resolution I think you could use one of the SHELXes (forgot the terminology) for refinement, that could be more appropriate. F.V. Deepthi wrote: Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi -- Deepthi -- Deepthi
[ccp4bb] Regarding refinement in Refmac5
Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi
Re: [ccp4bb] Regarding refinement in Refmac5
Thank You so much . I am trying to process the data again in all space groups possible for primitive hexagonal and try refinement again. And i didn't use Phenix after i got the MR solution. Probably refining the mutated model again would give some more information. Thank You once again. Appreciated Deepthi On Wed, Jul 18, 2012 at 10:02 AM, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: Can you check space group in your mtz and pdb? I have seen this happening when they disagree. It is annoying and I would like it to be sorted out. If you want you can send your data and I can try to sort it out. Garib On 18 Jul 2012, at 17:50, Deepthi wrote: I tried opening the model with other spacegroups MTZ file. The map doesn't fit well for other spacegroups. The initial model was refined using Phenix Autobuild software. I tried MR with every spacegroup possible in primitive hexagonal. Only p3221 worked. There is no twinning in the crystal. I will try using other softwares for refinement but this is annoying. I also tried mutating the model to poly alanines and refine but this made it worse. The R-free went up to 0.546. I initially thought it might be a space group problem but trying other space groups doesn't work either. Thank youvery much for the help Deepthi On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi there, Not much information provided. How was the initial model refined ? Phenix ? It could be a problem with the Refmac refinement protocol (difficult to say with so little information) if you switched from Phenix to Refmac. How certain are you 1 - of the space group; 2 - that the crystal wasn't twinned ? You can have both and it can be annoying. Further, at this resolution I think you could use one of the SHELXes (forgot the terminology) for refinement, that could be more appropriate. F.V. Deepthi wrote: Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi -- Deepthi Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk jen...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk -- Deepthi
Re: [ccp4bb] problem in scaling the Zn-MAD data
Hello I arrived at the p312 space group by running a self rotation function using MOLREP. The maps show the space group as p312. I was scaling the data individually for each wavelength. None of the three wavelengths are scaling are scaling in p312 space group. On Thu, Apr 5, 2012 at 2:17 AM, Clemens Vonrhein vonrh...@globalphasing.com wrote: Hi, On Wed, Apr 04, 2012 at 02:07:58PM -0700, Deepthi wrote: Hello everyone I have a problem scaling the MAD data which was collected a week ago.The data was collected at 1.5A resolution using three wavelengths for Zn-MAD experiments. Scaling the data for MAD experiments, the number of rejections and chi2 values were very high even after adjusting the error-scale factor and error model. The space group i used was p312 which i obtained by running a self-rotation function in MOLREP. When i scale my data using p312 spacegroup the chi2 and rejections were huge. But he data was scaling well in p321 spacegroup. can anyone explain whats going on? When you say 'Scaling the data for MAD experiments': do you mean scaling the various scans for your 3-wvl MAD data in a single scaling job? Unless you already took care of this during data integration, remember that your separate scans could have been indexed differently and therefore don't match up. See eg. http://www.ccp4.ac.uk/html/reindexing.html for some lookup-tables in P312 and P321. You can use the CCP4 program 'reindex' on MTZ files if needed. But I guess most modern data-processing and scaling programs will take care of that automatically anyway? Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) *** -- Deepthi
[ccp4bb] problem in scaling the Zn-MAD data
Hello everyone I have a problem scaling the MAD data which was collected a week ago.The data was collected at 1.5A resolution using three wavelengths for Zn-MAD experiments. Scaling the data for MAD experiments, the number of rejections and chi2 values were very high even after adjusting the error-scale factor and error model. The space group i used was p312 which i obtained by running a self-rotation function in MOLREP. When i scale my data using p312 spacegroup the chi2 and rejections were huge. But he data was scaling well in p321 spacegroup. can anyone explain whats going on? Thank you very much Deepthi
[ccp4bb] Using intrinsically bound Zn atoms for phasing
Hi I am trying to solve the structure of an engineered protein.The protein is crystallized with Zn bound to it .We collected a 1.5A0 data. Molecular Replacement didn't yield a good match for the protein. I want to try MAD taking advantage of the Zn atoms in protein. I am not sure what wavelength should i use to collect the diffraction data for Zn. any suggestions? Thank You Deepthi -- Deepthi
