Re: [ccp4bb] molprobity
It works now. Thanks for fixing. Sincerely,SDY Date: Mon, 11 Nov 2013 16:13:52 + From: tanne...@missouri.edu Subject: [ccp4bb] molprobity To: CCP4BB@JISCMAIL.AC.UK I encountered the same problem last night. The server won't upload the PDB file. I didn't check this morning. On Nov 11, 2013, at 10:02 AM, SD Y wrote: Hi, I was wondering if molprobity server is down (http://molprobity.biochem.duke.edu/index.php) or its just my files or our network has problem because I cant upload my files (some times it gave error). Thanks SDY John J. Tanner Professor of Biochemistry and Chemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 email: tanne...@missouri.edu phone: 573-884-1280 fax: 573-882-2754 web: http://www.chem.missouri.edu/tannergroup/tanner.html
[ccp4bb] molprobity
Hi, I was wondering if molprobity server is down (http://molprobity.biochem.duke.edu/index.php) or its just my files or our network has problem because I cant upload my files (some times it gave error). ThanksSDY
Re: [ccp4bb] low-resolution and zinc
I am using Refmac_5.7.0029 in CCP4ThanksSDY Date: Fri, 9 Nov 2012 20:35:48 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK What program do you use for refinement?FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 9, 2012, at 20:09 , SD Y wrote:Dear All, Thanks for all the suggestions on low resolution, SG and Zn and I learned a lot. I am not done yet. I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger Rowlett. Also an excellent protocol is available at http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. Protocol seems to be very straight forward. I am not getting the result I wanted. 1. When ever I generate cif file, its not writing any for ZN-SG links at all. 2. After refmac refinement it only draw in LINK for His-ZN. Please see the imageshttps://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.pnghttps://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png 3. I get this His-Zn link without using .cif file, so what stage do I use this .cif file? 4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing LINK in PDB. I only see LINKR ZNZN H 1 SG CYS A 83ZN-CYS I dont know what is missing, I also attached the log file which generated the ZN-His coordination. Any help is highly appreciated. ThanksSDY Date: Thu, 8 Nov 2012 14:36:59 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK The experiment should be very problematic if I can't determine point group on the base of the symmetry merging statistics.Watch CHI2 :-) Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com wrote:Then we agree. I got confused because you mentioned"space group" and not "point group" in your phrase about PHASER and MOLREP and was afraid others might have gotten confused as well. Also, in case of twinning or almost crystallographic non-crystallographic symmetry, determining the point group on the basis of processing statistics alone can be inconclusive or even misleading. If I recall correctly, there has recently been a thread about this in the bulletin board.Herman <74_refmac5_1.log>
[ccp4bb] low-resolution and zinc
Dear all, I have a related question to the one I have posted "low resolution and SG", on which I am still working based on the suggestions I have got. The model I have used, has Zn co-ordinated well in tetrahydral fashion by 3 cys and 1 His residues. They have add Zn in to their experiment. In my 3.4 A structure (I am still working on right SG), initial maps show very strong positive density (sigma=6.5) at the place of Zn ( https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have not used Zn in my experiment. I could only suspect Tryptone and yeast extract which I used to make media. I would like to know how likely this positive density belongs to Zn? How to reason the presence of Zn when its not been used?Is there is any way to confirm if its Zn. If this is not Zn, what else could it be? Any thing I could try to rule out or in Zn or other ions. I appreciate your help and suggestions. Sincerely, SDY
Re: [ccp4bb] protein cleavage
Rana, I understood that these proteases are very efficient in cleavage. One time, I had a construct MBP-3C-protein, I never able to cleave this particular construct while I could do well with other truncations.The MBP you are seeing might be co-purified with DHBx and unless gel band intensity suggest that its coming from fusion protein. I feel cleavage site may not be accessible for the protease. Might be adding few more residues might help. Good luckSDY Date: Sun, 4 Nov 2012 07:24:42 -0800 From: rna19792...@yahoo.com Subject: [ccp4bb] protein cleavage To: CCP4BB@JISCMAIL.AC.UK Dear CCP4 I am having a problem with cleaving my fusion protein and I would be grateful if you advice me regarding this situation, I have an MBP-DHBx fusion protein and I am trying to cleave it using TEV protease, I have tried different ratios and different temperatures with different incubation time but still it will not cleave, all I observe on the gel is the bands of the fusion protein which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa and no sign of the DHBx which is 17kDa,I have also checked the sequence if there was any problem but I could not find anything unusual the sequence was fine , so if you have any suggestions regarding this situation I will be thankful Best Regards Rana
[ccp4bb] low-resolution data and SG
Dear All, I have few basic questions for which I need help. I have a 3.4 A data and I have processed it to P4. 1. I used pointless to find SG, it suggests P41 21 2. But I see two strong intensities in systematic absences Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 2 -0.7 0.3 -2.0 0 0 3 1.0 0.4 2.3 0 0 5 0.3 0.7 0.4 0 0 6 -0.7 0.9 -0.8 0 0 7 -0.4 0.9 -0.4 0 0 9 -0.2 0.9 -0.2 0 0 10 1.3 1.2 1.1 0 0 11 -0.8 2.1 -0.4 0 0 13 1.2 2.1 0.6 0 0 14 2.3 1.8 1.3 0 0 15 -1.0 1.9 -0.5 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 3 0 0 -0.1 0.2 -0.8 5 0 0 0.2 0.2 0.9 7 0 0 -0.3 0.2 -1.3 9 0 0 0.0 0.5 0.0 11 0 0 -0.2 0.6 -0.4 13 0 0 0.8 0.7 1.1 15 0 0 -1.2 0.6 -1.9 17 0 0 -0.3 0.8 -0.4 19 0 0 -1.4 0.6 -2.6 21 0 0 -2.2 1.2 -1.9 23 0 0 -0.8 1.3 -0.6 25 0 0 -1.2 1.1 -1.1 27 0 0 -0.9 1.6 -0.5 29 0 0 -0.4 1.7 -0.2 31 0 0 -7.1 1.3 -5.3 33 0 0 -2.4 2.1 -1.1 2. When I used phaser for MR, it gave weak solution in p43, so I scaled data in p43 21 2 (this also two intesities high like above in systamatic absences) and used for Phaser to get the following solution SINGLE solution SOLU SET RFZ=4.5 TFZ=9.4 PAK=0 LLG=105 TFZ==10.1 RF++ TFZ=17.7 PAK=0 LLG=282 TFZ==15.6 LLG=285 TFZ==12.4 SOLU SPAC P 43 21 2 SOLU 6DIM ENSE ensemble1 EULER 153.1 50.3 73.2 FRAC -0.11 0.03 -0.94 BFAC -2.65 SOLU 6DIM ENSE ensemble1 EULER 148.4 129.9 252.8 FRAC -0.32 -0.35 1.07 BFAC 4.01 Ensemble ensemble1 RMS variance(s): 1.13 3. I used this solution to further refine the model in refmac, using local ncs, with/without jelly, optimized weight/weight of 0.03, map sharpening with B=20 in several rounds. I noticed that R factor R factor stayed around 33% while R free keeps floating around 42%. I could see some density for missing loop in the model and I could build but the R work and R free moving apart. By reading, I understand that this is very common for low resolution data unless I use appropriate restraints. I am wondering if my space group is correct? I had understood that if it’s right SG, high intensity reflections will not be found in systematic absences but I started doubting if I have understood correctly. This is my first low resolution data, I want use this opportunity to learn refmac well. So could you please let me know if my doubt is right regarding SG and how do I troubleshoot. Thanks SDY
