Re: [ccp4bb] ANODE anomalous map in pymol
Deart Tim and Kamel, Thank you for your valuable advice. As Tim pointed out, the Shelx2map worked perfectly fine, and these are indeed very useful tools for anyone who is moving the map from the Shelx to pymol or CCP4. Yes, i did try the COOT exported map extension to .ccp4, but it did not work for me. I do not know why. However, the exporting segmented map did the required job and can be easily open into the PYMOL. In addition, Shelx2map did a great job as you can now make a single map file for making the anomalous map for whole pdb in just one step. Thank you for the help and time. Appu On 1 March 2017 at 07:19, Kamel El Omari <kamel.el-om...@diamond.ac.uk> wrote: > Hi Appu, > > Did you change the file extension of the exported map extension to .ccp4 > (instead of .map)? That worked for me. > Cheers > Kamel > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Paul Emsley > Sent: 28 February 2017 22:57 > To: ccp4bb > Subject: Re: [ccp4bb] ANODE anomalous map in pymol > > On 28/02/2017 22:54, Appu kumar wrote: > > Hi, > > I Already did that in COOT, but PYMOL does not read it in map format. > > Pymol fail to show mesh in isomesh command. > > I confess that I don't know how PyMOL works. Perhaps Export Map Fragment > will do what you need. > > Paul. > > -- > This e-mail and any attachments may contain confidential, copyright and or > privileged material, and are for the use of the intended addressee only. If > you are not the intended addressee or an authorised recipient of the > addressee please notify us of receipt by returning the e-mail and do not > use, copy, retain, distribute or disclose the information in or attached to > the e-mail. > Any opinions expressed within this e-mail are those of the individual and > not necessarily of Diamond Light Source Ltd. > Diamond Light Source Ltd. cannot guarantee that this e-mail or any > attachments are free from viruses and we cannot accept liability for any > damage which you may sustain as a result of software viruses which may be > transmitted in or with the message. > Diamond Light Source Limited (company no. 4375679). Registered in England > and Wales with its registered office at Diamond House, Harwell Science and > Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom >
Re: [ccp4bb] ANODE anomalous map in pymol
Hi Paul and Clement, As suggested by the Paul, Export the map fragment from the COOT, did the trick. It worked perfectly fine. Thank for valuable suggestions, indeed helpful. Appu On 28 February 2017 at 19:55, Appu kumar <appu.kum...@gmail.com> wrote: > Hi, > Yes I tried renaming the file to .map, .ccp4, .map.xplor. Nothing seems > to be making mesh in pymol around the corresponding atom. > > Thank you for help and time > Appu > > On 28 February 2017 at 19:38, Clement Angkawidjaja <clem...@evec.jp> > wrote: > >> Have you tried changing the file extension to .ccp4 instead of .map? >> >> Cheers, >> Clement >> >> *From:* Appu kumar <appu.kum...@gmail.com> >> *Sent:* Wednesday, March 01, 2017 7:54 AM >> *To:* CCP4BB@JISCMAIL.AC.UK >> *Subject:* Re: [ccp4bb] ANODE anomalous map in pymol >> >> Hi, >> I Already did that in COOT, but PYMOL does not read it in map format. >> Pymol fail to show mesh in isomesh command. >> Thank you >> >> Appu >> >> On 28 February 2017 at 16:31, Paul Emsley <pems...@mrc-lmb.cam.ac.uk> >> wrote: >> >>> On 28/02/2017 20:44, Appu kumar wrote: >>> >>>> Dear CCP4 Users, >>>> I ran anode to calculate the anomalous map for heavy atoms in protein. >>>> ANODE output the >>>> anomalous map in .pha file, which can be viewed in COOT. However, I >>>> want to get the >>>> anomalous map from .pha file to .map or .xplor file, which can be feed >>>> into PYMOL to make >>>> maps. Is there a way to extract the anomalous map information from .pha >>>> file to .map or >>>> .xplor file. >>>> >>> >>> In Coot: >>> >>> File -> Export Map >>> >> >> > >
Re: [ccp4bb] ANODE anomalous map in pymol
Hi, Yes I tried renaming the file to .map, .ccp4, .map.xplor. Nothing seems to be making mesh in pymol around the corresponding atom. Thank you for help and time Appu On 28 February 2017 at 19:38, Clement Angkawidjaja <clem...@evec.jp> wrote: > Have you tried changing the file extension to .ccp4 instead of .map? > > Cheers, > Clement > > *From:* Appu kumar <appu.kum...@gmail.com> > *Sent:* Wednesday, March 01, 2017 7:54 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] ANODE anomalous map in pymol > > Hi, > I Already did that in COOT, but PYMOL does not read it in map format. > Pymol fail to show mesh in isomesh command. > Thank you > > Appu > > On 28 February 2017 at 16:31, Paul Emsley <pems...@mrc-lmb.cam.ac.uk> > wrote: > >> On 28/02/2017 20:44, Appu kumar wrote: >> >>> Dear CCP4 Users, >>> I ran anode to calculate the anomalous map for heavy atoms in protein. >>> ANODE output the >>> anomalous map in .pha file, which can be viewed in COOT. However, I want >>> to get the >>> anomalous map from .pha file to .map or .xplor file, which can be feed >>> into PYMOL to make >>> maps. Is there a way to extract the anomalous map information from .pha >>> file to .map or >>> .xplor file. >>> >> >> In Coot: >> >> File -> Export Map >> > >
Re: [ccp4bb] ANODE anomalous map in pymol
Hi, I Already did that in COOT, but PYMOL does not read it in map format. Pymol fail to show mesh in isomesh command. Thank you Appu On 28 February 2017 at 16:31, Paul Emsley <pems...@mrc-lmb.cam.ac.uk> wrote: > On 28/02/2017 20:44, Appu kumar wrote: > >> Dear CCP4 Users, >> I ran anode to calculate the anomalous map for heavy atoms in protein. >> ANODE output the >> anomalous map in .pha file, which can be viewed in COOT. However, I want >> to get the >> anomalous map from .pha file to .map or .xplor file, which can be feed >> into PYMOL to make >> maps. Is there a way to extract the anomalous map information from .pha >> file to .map or >> .xplor file. >> > > In Coot: > > File -> Export Map >
[ccp4bb] ANODE anomalous map in pymol
Dear CCP4 Users, I ran anode to calculate the anomalous map for heavy atoms in protein. ANODE output the anomalous map in .pha file, which can be viewed in COOT. However, I want to get the anomalous map from .pha file to .map or .xplor file, which can be feed into PYMOL to make maps. Is there a way to extract the anomalous map information from .pha file to .map or .xplor file. I appreciate any incoming suggestions and comments. Thanks in advance Appu
Re: [ccp4bb] ANISOU in pdb and density improvement
Hello All, Sorry for giving incomplete information. The protein is 75KDa membrane protein and it exists as tetramer in ASU. Structure is solved by MR. Overall completeness of data is 98% wiith multiplicity of 4.8. Density looks great after refinement having ANISOU record in PDB. Any suggestions and guidance will be much appreciated Thanks you Appu On 1 July 2015 at 21:14, xaravich ivan xaravich.i...@gmail.com wrote: 4 Angstrom resolution is pretty low and there has to be other info associated with that to get more help from here. How big is your protein? How are you solving the phases? How complete is your data at that resolution? What kind of multiplicity are you getting? I think you have other issues that are much more pressing than what you are asking here!!! As you are able to get 4A data it is evident that you can crystallize your protein. Congrats Now, concentrate on that and try to get higher resolution data from better diffracting crystals and it might be a much easier experience, (unless you trying to solve a huge ribosomal complex kind of protein and in that case 4 angs is amazing) cheers Ivan On Wed, Jul 1, 2015 at 5:57 PM, Appu kumar appu.kum...@gmail.com wrote: Dear CCP4 users, I am refining a structure at 4A resolution. Crystal diffracted anisotropically and after refinement in both PHENIX and REFMAC, electron density in one of the domain of protein is represented by discontinuity and poor maps. Therefore i did the aniosotropy correction using anisotropy server. After anisotropy correction using anisotropy server, the electron density becomes resonable for 4A map as evident by appearance of electron density around some bulky sidechains and discontinuity in electron density is diminished. When i look into the pdb, I found that pdb contain ANISOU records for each atoms. I tried same things with other data sets and same data set again (data set described above) of same protein. After refinement, the ANISOU records are not present for each atoms in refined pdb and the electron density is discontinuous and poor. I am wondering why one of the anisotropically corrected MTZ by diffraction server gives the ANISOU records with the good density after refinement and other mtz file do not produce ANISOU record in pdb and poor density map. I would appreciate your help regarding this issue. Thank you Appu
[ccp4bb] ANISOU in pdb and density improvement
Dear CCP4 users, I am refining a structure at 4A resolution. Crystal diffracted anisotropically and after refinement in both PHENIX and REFMAC, electron density in one of the domain of protein is represented by discontinuity and poor maps. Therefore i did the aniosotropy correction using anisotropy server. After anisotropy correction using anisotropy server, the electron density becomes resonable for 4A map as evident by appearance of electron density around some bulky sidechains and discontinuity in electron density is diminished. When i look into the pdb, I found that pdb contain ANISOU records for each atoms. I tried same things with other data sets and same data set again (data set described above) of same protein. After refinement, the ANISOU records are not present for each atoms in refined pdb and the electron density is discontinuous and poor. I am wondering why one of the anisotropically corrected MTZ by diffraction server gives the ANISOU records with the good density after refinement and other mtz file do not produce ANISOU record in pdb and poor density map. I would appreciate your help regarding this issue. Thank you Appu
Re: [ccp4bb] CCP4 installation problem
Hello, I have already edited my .cshrc file where i removed the line which was pointing to older version and sourced the newer version ccp4-6.5/ccp4.setup-csh but problem still exist. Thank you Appu On 16 June 2015 at 09:25, Roger Rowlett rrowl...@colgate.edu wrote: Appu, You will have to edit your .tcshrc (startup) file to point to the correct setup script with a line like this: source /usr/local/xtal/ccp4-6.5/ccp4.setup-csh If you are using a different shell, e.g. bash, you will have to edit the appropriate startup file, e.g. .bashrc. You will likely find the offending line pointing to the old installation in your run command (startup) file and can edit it. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 6/16/2015 11:13 AM, Appu kumar wrote: Hello, I have downloaded 'ccp4-6.5 and deleted the older version of ccp4 installed. After installing the new version, it is giving the following problem on executing the ccp4i command. Error in startup script: couldn't read file /usr/local/xtal/ccp4-6.2.0/share/dbccp4i/ClientAPI/dbClientAPI.tcl: no such file or directory while executing source [FileJoin [GetEnvPath DBCCP4I_TOP] ClientAPI dbClientAPI.tcl] (file /home/appu/software/destination/ccp4-6.5/share/ccp4i/src/projectdirs.tcl line 23) invoked from within source [SearchPath TOP src projectdirs.tcl] (file /home/appu/software/destination/ccp4-6.5/share/ccp4i/src/system.tcl line 3379) invoked from within source [file join $env(CCP4I_TOP) src system.tcl] (file /home/appu/software/destination/ccp4-6.5/share/ccp4i/bin/ccp4i.tcl line 79) invoked from within source [file join $env(CCP4I_TOP) bin ccp4i.tcl] (file /home/appu/software/destination/ccp4-6.5/bin/ccp4i line 12) I have deleted ccp4-6.2.0 version. Somehow ccp4-6.5 is looking for the older version upon execution. I will be thankful your your advice. Appu
[ccp4bb] CCP4 installation problem
Hello, I have downloaded 'ccp4-6.5 and deleted the older version of ccp4 installed. After installing the new version, it is giving the following problem on executing the ccp4i command. Error in startup script: couldn't read file /usr/local/xtal/ccp4-6.2.0/share/dbccp4i/ClientAPI/dbClientAPI.tcl: no such file or directory while executing source [FileJoin [GetEnvPath DBCCP4I_TOP] ClientAPI dbClientAPI.tcl] (file /home/appu/software/destination/ccp4-6.5/share/ccp4i/src/projectdirs.tcl line 23) invoked from within source [SearchPath TOP src projectdirs.tcl] (file /home/appu/software/destination/ccp4-6.5/share/ccp4i/src/system.tcl line 3379) invoked from within source [file join $env(CCP4I_TOP) src system.tcl] (file /home/appu/software/destination/ccp4-6.5/share/ccp4i/bin/ccp4i.tcl line 79) invoked from within source [file join $env(CCP4I_TOP) bin ccp4i.tcl] (file /home/appu/software/destination/ccp4-6.5/bin/ccp4i line 12) I have deleted ccp4-6.2.0 version. Somehow ccp4-6.5 is looking for the older version upon execution. I will be thankful your your advice. Appu
[ccp4bb] Refinement at 4A resolution
Dear CCP4 Member, I seek your advice on the refinement issues at the low resolution 4A. I am trying to refine a membrane protein structure after getting the phases from MR using the PHASER. The soluble domain structure which comprises of 40% of protein has been used as template (sequence identity 80%) in MR search . The PHASER gave a good solution having TFZ value of about 14.3. I have then created the polyA model for the transmembrane domain from distant homolog which share 30% sequence identity for TM region and try to find the phases for whole TM domain keeping the soluble domain fixed. I got lucky in getting the phases for the whole protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork and Rfree got stalled at the 41 and 44 respectively after several cycle of the refinement in both refmac and phenix. I checked the spacegroup with pointless and it suggests C2221. I have attached the pointless and phenix.xtriage run file with this mail for your evaluation. Phenix.xtriage suggests no major pathologies with the data except the mild psuedomerohedral twining. There are two molecules of protein in ASU. Evaluation of the density maps, suggest reasonable map for the most of protein part. I am wondering why Rwork and Rfree are not coming down despite of the good MR solution and what i am doing wrong with refinement and if there is some pathologies associated with the data which needs to be answered before heading to refinement. Thanks for your help in advance. Appu 93_pointless.log Description: Binary data xtriage_50.log Description: Binary data
Re: [ccp4bb] Refinement at 4A resolution
Dear All, Sorry for the wrong pointless file. With this mail i have attached the pointless run file from the unmerged data. This file also suggests the C2221 spacegroup. Appu On 22 April 2015 at 17:40, Christian Roth christian.r...@bbz.uni-leipzig.de wrote: Hi Appu, you start already with a fixed spacegroup (scaled merged data) according to your pointless log. So you can't get another possible solution from pointless. Cheers Am 22.04.2015 um 22:28 schrieb Appu kumar: Dear CCP4 Member, I seek your advice on the refinement issues at the low resolution 4A. I am trying to refine a membrane protein structure after getting the phases from MR using the PHASER. The soluble domain structure which comprises of 40% of protein has been used as template (sequence identity 80%) in MR search . The PHASER gave a good solution having TFZ value of about 14.3. I have then created the polyA model for the transmembrane domain from distant homolog which share 30% sequence identity for TM region and try to find the phases for whole TM domain keeping the soluble domain fixed. I got lucky in getting the phases for the whole protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork and Rfree got stalled at the 41 and 44 respectively after several cycle of the refinement in both refmac and phenix. I checked the spacegroup with pointless and it suggests C2221. I have attached the pointless and phenix.xtriage run file with this mail for your evaluation. Phenix.xtriage suggests no major pathologies with the data except the mild psuedomerohedral twining. There are two molecules of protein in ASU. Evaluation of the density maps, suggest reasonable map for the most of protein part. I am wondering why Rwork and Rfree are not coming down despite of the good MR solution and what i am doing wrong with refinement and if there is some pathologies associated with the data which needs to be answered before heading to refinement. Thanks for your help in advance. Appu #CCP4I VERSION CCP4Interface 2.2.1 #CCP4I SCRIPT LOG pointless #CCP4I DATE 22 Apr 2015 17:39:30 #CCP4I USER appu #CCP4I PROJECT AS015crv6ctd2nq #CCP4I JOB_ID 119 #CCP4I SCRATCH /tmp/appu #CCP4I HOSTNAME sasha #CCP4I PID 47982 ### ### ### ### CCP4 6.4: POINTLESS version 1.9.16 : 21/08/14## ### User: appu Run date: 22/ 4/2015 Run time: 17:39:30 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. Input command lines HKLIN /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/imosflm/AS115c_1_0001.mtz HKLOUT /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/refine/AS115c_1_0001_pointless1.mtz ## This script run with the command ## # /home/appu/Downloads/ccp4-6.4.0/bin/pointless End of input OS type: linux Release Date: 21st August2014 ** ** * POINTLESS * * 1.9.16 * ** * Determine Laue group from unmerged intensities * * Phil Evans MRC LMB, Cambridge * * Uses cctbx routines by Ralf Grosse-Kunstleve et al.* ** ** Spacegroup information obtained from library file: Logical Name: SYMINFO Filename: /home/appu/Downloads/ccp4-6.4.0/lib/data/syminfo.lib Reflection list generated from file: /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/imosflm/AS115c_1_0001.mtz Title: Untitled Space group from HKLIN file : C 2 2 21 Cell: 130.42 213.36 160.80 90.00 90.00 90.00 Resolution range in file: 65.213.75 Time for reading file(s):2.190 secs === * Summary of test data read in: Resolution range accepted:65.213.75 Number of reflections = 23371 Number of observations =150865 Number of parts=619082 Number of batches in file = 600 Number of datasets = 1 Project: New Crystal: New Dataset: New Run number: 1 consists of batches 1 to600 Resolution range for run:65.213.75 Phi range: 0.00 to 180.00 Time range: 0.00 to 180.00 Closest reciprocal axis to spindle
Re: [ccp4bb] fastening crystal formation
Hello, If the homologous protein structure is available, or you have symmetry information of your crystal, then try engineering surface by putting in residues which has less entropy like replacing Lys to Arg, Met to Gln. We are able to fasten the crystal growth from 20 days to 3-4 days by creating phenylalanine Pi-stack. This is tricky but if you have an idea of crystal contact, then it should be straightforward without much trouble. Good Luck Appu On 31 October 2014 11:43, Patrick Shaw Stewart patr...@douglas.co.uk wrote: Michael (and some others) You haven't mentioned - and I guess don't use regularly - the random MMS approach, where crushed seed-crystals are added to random screens. This really is a terrific method, and it will on average roughly double productivity. It's the first thing I would think of in a case like Vijaykumar's (as I told him this morning!). Galina Obmolova and her colleagues published a great paper earlier this year about MMS. They were working with a set of 16 Fab fragments that comprised all combinations of 4 different heavy chains and 4 different light chains. Three structures were generated without MMS, but by various very creative uses of microseeding they were able to get all 16/16 structures. Ref below. Best wishes, Patrick __ Obmolova, G., Malia, T. J., Teplyakov, A., Sweet, R. W., Gilliland, G. L. (2014). Protein crystallization with microseed matrix screening: application to human germline antibody Fabs. *Structural Biology and Crystallization Communications*, *70*(8). On 31 October 2014 16:07, R. M. Garavito rmgarav...@gmail.com wrote: Although three months is a long time, it is no completely unheard of, and does not require the invocation of proteolysis. The longest time I have heard of is ~1 yr, so count yourself lucky. However to get good advice, as well as to use it, you need to ask yourself several questions: 1. What kind of crystals are they? Protein, salt, etc? If they are salt, don't pursue this condition. 2. How many crystals did you get? One or 2 in a drop or a microcrystal shower. And of what kind? Single, well shaped, rosettes, needle clusters, or something that looks crystalline. Screen more broadly around your initial hit. 3. How many times have your tried to repeat this? Once, twice, or more? Did you try setups in duplicate? If so, did you get reproducible results? Have you actually screened around these conditions, varying each component systematically (PEG, salt, pH, buffer, etc.)? 4. What method did you use? And in what kind of container? For one thing, we don't completely trust the integrity of our setups for longer than 2 months. All containers leak water slowly, so when crystals take longer than 2 months to grow (a) the real conditions are at much higher values than you naively think (i.e., the drop has dried out more than you expected) or (b) other components are crystallizing, for example a zinc salt. It depends what else is in your protein buffer, as well. To quicken protein crystallization (which is not always a good thing), increase your protein concentration (by 1.5-2x) and/or PEG concentration (such as screening up to 40% PEGmme 550). Sadly, crystallization is a combination of thermodynamic and kinetic factors: you can get crystals (sometimes a single crystal only) when just outside the truly optimal conditions, but this may be only a sporadic event. You got to keep screening. Good luck, Michael ** *R. Michael Garavito, Ph.D.* *Professor of Biochemistry Molecular Biology* *603 Wilson Rd., Rm. 513* *Michigan State University * *East Lansing, MI 48824-1319* *Office:* *(517) 355-9724 %28517%29%20355-9724 Lab: (517) 353-9125 %28517%29%20353-9125* *FAX: (517) 353-9334 %28517%29%20353-9334 Email: rmgarav...@gmail.com garav...@gmail.com* ** On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri vijaypkuma...@gmail.com wrote: Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975 -- 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
Re: [ccp4bb] Normal mode refinement
Dear All, Thank you very much for valuable suggestions and educating me on the normal mode refinement. Actually, I am trying to refine a protein (cytosolic domain and trans-membrane domain). I found a solution through PHASER and density looks really good in both domain but as i proceeds with refinement density remain great in both domain till Rfree around 38%. Interestingly, with further refinement cycle, Rfree reduced to 30% but the density in the trans-membrane domain becomes very weak. That is why i am wondering whether it is possible to improve the density in the trans-membrane domain by using Normal mode refinement. Conservatively speaking, it could be possible that trans-membrane is highly flexible or disordered and after much cerebration, i am thinking to incorporate the normal mode refinement to monitor if there is any improvement in electron density trans-membrane domain. I would follow suggestions of Dr, Mande and Dr. Ethan. Also, would give a try to what Arpita has suggested. I further, warmly welcome any suggestion on refinement procedure to improve electron density in flexible or disordered trans-membrane domain. Appu On 20 October 2014 23:41, Arpita Goswami bt.arp...@gmail.com wrote: Hello, You can also contact elNemo or NOMAD-Ref server developers about getting covariance/correlation matrices from normal mode analysis outputs to know the correctly coordinated mobile atoms. In this way you can compare with biological data also. In Shekhar's said paper K. Suhre (one of the developer of el-Nemo server) has done the same very correctly. best wishes, Arpita On Tue, Oct 21, 2014 at 5:40 AM, Appu kumar appu.kum...@gmail.com wrote: Dear CCP4 Users, I seek your valuable advice and suggestion in carrying out the normal mode structure refinement which manifest the dynamics of protein as linear combination of harmonic modes, used to describe the motion of protein structure in collective fashion. Studies suggest that it is highly useful in refining the protein structure which harbors a considerable magnitude of flexibility in atomic position owing to high thermal factors. Therefor I want to know is there any software/script available to execute the normal mode of refinement. Thanks a lot in advance for your imperative suggestions Appu -- Arpita -- Arpita Goswami Senior Research Fellow Structural Biology Laboratory Centre for DNA Fingerprinting and Diagnostics (CDFD) Tuljaguda (Opp MJ Market), Nampally, Hyderabad 500 001 INDIA Phone: +91- 40- 24749401/404 Mobile: 9390923667, 9502389184 Email: arp...@cdfd.org.in
Re: [ccp4bb] Normal mode refinement
Dear All, Thank you very much for valuable suggestions and educating me on the normal mode refinement. Actually, I am trying to refine a protein (cytosolic domain and trans-membrane domain). I found a solution through PHASER and density looks really good in both domain but as i proceeds with refinement density remain great in both domain till Rfree around 38%. Interestingly, with further refinement cycle, Rfree reduced to 30% but the density in the trans-membrane domain becomes very weak. That is why i am wondering whether it is possible to improve the density in the trans-membrane domain by using Normal mode refinement. Conservatively speaking, it could be possible that trans-membrane is highly flexible or disordered and after much cerebration, i am thinking to incorporate the normal mode refinement to monitor if there is any improvement in electron density trans-membrane domain. I would follow suggestions of Dr, Mande and Dr. Ethan. Also, would give a try to what Arpita has suggested. I further, warmly welcome any suggestion on refinement procedure to improve electron density in flexible or disordered trans-membrane domain. Appu On 21 October 2014 07:39, Appu kumar appu.kum...@gmail.com wrote: Dear All, Thank you very much for valuable suggestions and educating me on the normal mode refinement. Actually, I am trying to refine a protein (cytosolic domain and trans-membrane domain). I found a solution through PHASER and density looks really good in both domain but as i proceeds with refinement density remain great in both domain till Rfree around 38%. Interestingly, with further refinement cycle, Rfree reduced to 30% but the density in the trans-membrane domain becomes very weak. That is why i am wondering whether it is possible to improve the density in the trans-membrane domain by using Normal mode refinement. Conservatively speaking, it could be possible that trans-membrane is highly flexible or disordered and after much cerebration, i am thinking to incorporate the normal mode refinement to monitor if there is any improvement in electron density trans-membrane domain. I would follow suggestions of Dr, Mande and Dr. Ethan. Also, would give a try to what Arpita has suggested. I further, warmly welcome any suggestion on refinement procedure to improve electron density in flexible or disordered trans-membrane domain. Appu On 20 October 2014 23:41, Arpita Goswami bt.arp...@gmail.com wrote: Hello, You can also contact elNemo or NOMAD-Ref server developers about getting covariance/correlation matrices from normal mode analysis outputs to know the correctly coordinated mobile atoms. In this way you can compare with biological data also. In Shekhar's said paper K. Suhre (one of the developer of el-Nemo server) has done the same very correctly. best wishes, Arpita On Tue, Oct 21, 2014 at 5:40 AM, Appu kumar appu.kum...@gmail.com wrote: Dear CCP4 Users, I seek your valuable advice and suggestion in carrying out the normal mode structure refinement which manifest the dynamics of protein as linear combination of harmonic modes, used to describe the motion of protein structure in collective fashion. Studies suggest that it is highly useful in refining the protein structure which harbors a considerable magnitude of flexibility in atomic position owing to high thermal factors. Therefor I want to know is there any software/script available to execute the normal mode of refinement. Thanks a lot in advance for your imperative suggestions Appu -- Arpita -- Arpita Goswami Senior Research Fellow Structural Biology Laboratory Centre for DNA Fingerprinting and Diagnostics (CDFD) Tuljaguda (Opp MJ Market), Nampally, Hyderabad 500 001 INDIA Phone: +91- 40- 24749401/404 Mobile: 9390923667, 9502389184 Email: arp...@cdfd.org.in
[ccp4bb] Normal mode refinement
Dear CCP4 Users, I seek your valuable advice and suggestion in carrying out the normal mode structure refinement which manifest the dynamics of protein as linear combination of harmonic modes, used to describe the motion of protein structure in collective fashion. Studies suggest that it is highly useful in refining the protein structure which harbors a considerable magnitude of flexibility in atomic position owing to high thermal factors. Therefor I want to know is there any software/script available to execute the normal mode of refinement. Thanks a lot in advance for your imperative suggestions Appu
[ccp4bb] Refinement problem
Hello All, I am new to low resolution refinement parametrization and regularization. Crystal diffracted with high anisotropy reaching to 3.5A in one direction and 4.5A other direction. I am refining a structure at 3.9A resolution. Protein has two domain connected trhough a linker and is packed as tetramer in ASU. Refinement in phenix as well as in refmac leads to wipe away of electron density in helical domain of protein leaving only blobs of electron density while other domain have good amount of density after refinement. I need your precious and valuable suggestion for proceeding with refinement. Thanks in advance for your suggestion. Thank you
[ccp4bb] CNS MR Help
Hello All, I have tried MR in Phaser, MRage, Molrep , Mrbump but i am not getting the true solution which it supposed to be. Although the resoution of data is 6.1A, but i want to give a try to CNS and see if I can find right solution. I have read the manual of CNS but i am unable to get the CNS running. Would you please help me in running the CNS program. Thank you Appu
[ccp4bb] Help in getting source of HOLE programme
Hello everyone, I request all of you to please help in getting the source of hole programme which is required to visualize the cavity running through the channel in protein structures. This programme is written by Dr. Oliver Smart. Despite of exhaustive search on various place on web, I am unable to find the source of this programme and therefor request to you, if any one has the source or link to get this progammes please provide me. I will be highly thank full for the help. Thank you Appu
Re: [ccp4bb] Orientation of molecules
Dear All, I think i have not explained my problem precisely. This may be weird one but let me elaborate more. I have have a protein moleculeA, having N-term, and C-term end. Structurally, it is dimer with anti-parallel arrangement i.e N-terminal of one copyA of molecule form dimer in such a way that it copyB would be arranged in antiparallel fashioned (N-term of copyA is besides C-term of CopyB). So when i am searching for two copy of molecule in phaser it is giving me two copy of molecule in parallel arrangement. So my question is, how to tell phaser that after fixing the orientation of first copy, to change the orientation of 2nd copy with respect to first one so that their n-teminal and c-terminal lies beside each other. I am looking for your valuable suggestion. Thank you On 21/11/2013, Matthias Zebisch matth...@strubi.ox.ac.uk wrote: i think you need to explain the problem in greater detail to the community eg. how do you come to know that the orientation of mol2 is 180° flipped - perhaps it is correct as phaser puts it? No one will be able to help you with so little information Best wishes, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 11/21/2013 5:38 PM, Appu kumar wrote: Dear All, I seek your valuable suggestion on a MR problem. I am asking phaser to search for two molecules in ASU, first molecules phaser searched right but when placing the second molecule, it orientation has got flipped by 180 deegree. Is there any way to tell phaser to fix the orientation of second copy of molecules. ? Looking forward for your valuable suggestion Thank you very much in advance Appu -- - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk -
[ccp4bb] Orientation of molecules
Dear All, I seek your valuable suggestion on a MR problem. I am asking phaser to search for two molecules in ASU, first molecules phaser searched right but when placing the second molecule, it orientation has got flipped by 180 deegree. Is there any way to tell phaser to fix the orientation of second copy of molecules. ? Looking forward for your valuable suggestion Thank you very much in advance Appu
Re: [ccp4bb] expanding reflections from C2221 to P21
How do you suspect that C2221 is 'pseudo' and P21 is 'real'? You can use the reindex programme incorporated in ccp4 suit. Reindex programme can expand symmetry from C2221 to P21.I Hope you will get the result. Thank you Appu On 2 October 2013 21:43, wtempel wtem...@gmail.com wrote: Hello, I would like to expand a reflection data set in mtz format from C2221 to P21. The purpose is to obtain consistent R-free flags based on a structure already refined in C2221 for a related data set that I suspect is pseudo-C2221 but real P21. Primitive cell dimensions are: 37.6 126.1 40.61 89.99 117.6 90.01, C-centered: 37.6 71.99 126.1 90 89.99 90.01 pointless provides the following matrix: pointless Reindex operator from input cell to lattice cell: [h,h+2l,-k] h' = ( h k l ) ( 1 1 0 ) ( 0 0 -1 ) ( 0 2 0 ) /pointless In sftools, I loaded the C2221 data set and did sftools$ reindex matrix 1 0 0 0 0 -1 -.5 .5 0 with the transposed (to account for the presumably inverted order of factors in the program?) inverse matrix of the one listed above with the aim of restoring the primitive asymmetric unit. I was encouraged seeing sftools report new cell dimensions matching the expected primitive cell. Then I did sftools$ expand 4 I expected now to have a workable P21 version of my C2221 data set, but molecular replacement (MOLREP) with my C2221 model failed to place even a single copy of the model. Thus, I must have misused sftools by issuing commands that were either wrong or in the wrong order or my application of linear algebra was mistaken. Any ideas out there? Thanking you in advance, Wolfram Tempel
Re: [ccp4bb] off topic: Fluorescence labeled protein, neutral at pH=7
Deal Xiang, You can think of GFP/YFP protein attached with any protein would be good for your experiment. These can be easily cloned and expressed. Alternatively one more approach is mentioned below. Chose a protein which has only one cysteine, that too at the surface then you can think of labelling your protein with thiol-ester of commerically available atto-dyes. You can also think of mutating alanine present at the surface with cysteine which subsequently can be covalently attached with the thio-reactinve dyes. It is two step process, first you have to set up a chemical reaction linking the thiol mediated covalent ligation of dyes to your protein, then further purifying the lablled protein with HPLC. On 17 September 2013 00:54, 李翔 lixiang1...@gmail.com wrote: Hi everyone, I want to get some fluorescence labeled protein for my experiment. I wish it is under 40K Da and have isoelectric point of 7 or slightly lower. Do you have any suggestions on the commercially available protein please? Thanks very much for your help! Sincerely, Xiang -- Li Xiang Department of chemistry, Purdue University Email：lixiang1...@gmail.com
[ccp4bb] Double stranded DNA concentration estimation
Deal CCP4 member's Sorry for asking Off topic question. Please correct me if i am doing wrong measurements. I am determining the double stranded concentration using the absorbance spectrophotometer reading at 260A and extinction coefficients for individual NMPs . But protein to DNA stoichiometry is coming really off in ITC. I am doubting weather i am using right method to determine the DNA concentration accurately or i am doing some wrong calculation. I am calculating the DNA extinction coefficients by simply multiplying the total number of individual nucleotide by it extinction coefficients which are as follow A = 15.4 mM-1 cm-1 C = 7.4 mM-1 cm-1 G = 11.8 mM-1 cm-1 T = 9.6 mM-1 cm-1 The formula which i am using to calculate the concentration is DNA concentration(M)= ( Abs@260-Abs@330)/Molar extinction coefficients of DNA I am multiplying this by dilution factor.
Re: [ccp4bb] STRAP secondary structure
Dear John, You can you the Prosmart http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/prosmart/documentation.html and also pdbsum can give what you asking for. Thanks Appu On 27 April 2013 02:57, Mike John perturb-w...@hotmail.com wrote: Hello, Every one, I am preparing a ppt presentation in an emergency style. What I want is a graph of sequence alignment with secondary structure on the top. Using STRAP i can got alignment nicely, but did not know the tips to add secondary structure on top. Anybody of experienced can give me a hand? Tutorial-like instruction would be much helpful. Your kindness is greatly appreciated. Thanks Mike
Re: [ccp4bb] molecular replacement problem.
Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies (not just two copies but also one copy and three copies just in case you have one of the extreme cases of solvent content)? (2) If the MR solution is correct and there is physical room for a DNA binding domain in your lattice (check by displaying symmetry mates), perhaps the DNA binding domain is disordered. In that case (and if all attempts with current data fail), you may have to crystallize the protein in presence of DNA. Good luck! Raji On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.com wrote: Dear members, I am doing a molecular replacement of a transcription factor whose ligand binding structure(24000 Da) is available in PDB but not for the DNA binding(13000 Da). When i am searching for the two copies from ligand binding domain as a template model, i am getting very good solution but i am not getting any density for the DNA binding domain to build up in density. The space gorup is P 1 21 1 (4) and unit cell parameters are Unit Cell: 57.43 69.36 105.99 90.00 90.00 90.00. Please guide me how to get the complete model structure. Table below show the matthews statistics For estimated molecular weight 37000. Nmol/asym Matthews Coeff %solvent P(2.20) P(tot) _ 1 5.7178.46 0.00 0.01 2 2.8556.91 0.62 0.70 3 1.9035.37 0.37 0.29 4 1.4313.82 0.00 0.00 _ The phaser molecular replacement gives the following table. istogram of relative frequencies of VM values -- Frequency of most common VM value normalized to 1 VM values plotted in increments of 1/VM (0.02) --- relative frequency --- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ||||||||||| 10.00 - 8.33 - 7.14 - 6.25 - 5.56 - 5.00 - 4.55 - 4.17 - 3.85 -- 3.57 --- 3.33 -- 3.12 -- 2.94 (COMPOSITION*1) 2.78 --- 2.63 2.50 - 2.38 2.27 -- 2.17 --- 2.08 -- 2.00 -- 1.92 --- 1.85 --- 1.79 --- 1.72 - 1.67 - 1.61 - 1.56 - 1.52 - 1.47 * (COMPOSITION*2) 1.43 - 1.39 - 1.35 - 1.32 - 1.28 - 1.25 - $TABLE : Cell Content Analysis: $SCATTER :N*Composition vs Probability:0|3x0|1:1,2: $$ N*Composition Probability $$ loggraph $$ 1 0.306066 2 0.00141804 $$ Most probable VM for resolution = 2.27817 Most probable MW of protein in asu for resolution = 92664.2 Thank a lot in advance -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] molecular replacement problem.
I run the phenix.xtriage to evaluate the twining but it suggest no twining. When i reindex from C2221 to P21, the completeness of data reduced from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2 resolution. I do not understand why the completeness of data reduced so much on reindexing. please Can anyone explain this phenomenon. Thank you On 24 March 2013 13:30, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de wrote: the p21 c2221 ambivalence can mean severe twinning (i had a similar case just now - try several crystals from the same condition) ! What do the twinning statistics suggest? cheers, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/24/2013 7:46 AM, Appu kumar wrote: Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies (not just two copies but also one copy and three copies just in case you have one of the extreme cases of solvent content)? (2) If the MR solution is correct and there is physical room for a DNA binding domain in your lattice (check by displaying symmetry mates), perhaps the DNA binding domain is disordered. In that case (and if all attempts with current data fail), you may have to crystallize the protein in presence of DNA. Good luck! Raji On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.comwrote: Dear members, I am doing a molecular replacement of a transcription factor whose ligand binding structure(24000 Da) is available in PDB but not for the DNA binding(13000 Da). When i am searching for the two copies from ligand binding domain as a template model, i am getting very good solution but i am not getting any density for the DNA binding domain to build up in density. The space gorup is P 1 21 1 (4) and unit cell parameters are Unit Cell: 57.43 69.36 105.99 90.00 90.00 90.00. Please guide me how to get the complete model structure. Table below show the matthews statistics For estimated molecular weight 37000. Nmol/asym Matthews Coeff %solvent P(2.20) P(tot) _ 1 5.7178.46 0.00 0.01 2 2.8556.91 0.62 0.70 3 1.9035.37 0.37 0.29 4 1.4313.82 0.00 0.00 _ The phaser molecular replacement gives the following table. istogram of relative frequencies of VM values -- Frequency of most common VM value normalized to 1 VM values plotted in increments of 1/VM (0.02) --- relative frequency --- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ||||||||||| 10.00 - 8.33 - 7.14 - 6.25 - 5.56 - 5.00 - 4.55 - 4.17 - 3.85 -- 3.57 --- 3.33 -- 3.12 -- 2.94 (COMPOSITION*1) 2.78 --- 2.63 2.50 - 2.38 2.27
Re: [ccp4bb] molecular replacement problem.
Sorry for the misconception. Yes i am expanding the space group from merged mtz file. Actually i have enough number of images collected. when i indexed, integrate, and scale the data in either C2221 or P 21, it fetches the overall 98% completeness. But when i am trying to reindex the data from C2221 to P21 keeping the Rfree flag of C2221, the completeness of data reduced drastically to 40%. This is what i am not getting. I am a beginner so i have to read a lot which i am doing also, but i had few practical confusion which i shared and off course i am getting good response. Thank you all for your kind response and educating me on the problem i faced. Thank you all for your valuable response. On 24 March 2013 15:15, vellieux frederic.velli...@ibs.fr wrote: Hello, Here we deal with symmetry and the unique part of reciprocal space (the reciprocal space asymmetric unit so to speak). C222(1) has eight asymmetric units (international tables, space group 20); P2(1) only has two. Assuming that Friedel's law does apply, then the minimum rotation range to collect a non-redundant data set (one observation per reflection) is 90 degrees, provided that the crystal is correctly and perfectly aligned. Normally with our current data collection methods where the crystal is randomly oriented, we would collect more than 90 degrees (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR beamline where you cannot really check during data collection how well the crystal fares during exposure to the X-rays - shoot first, think later. The reciprocal space asymmetric unit in C222(1) is smaller. I assume that what you are doing is to take the reduced data set file (an MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will not cover the monoclinic reciprocal space asymmetric unit by doing so. The way to do it is to take the file from processing, before (crystallographic symmetry) merging of the equivalents, and perform the scaling and merging in the P2(1) space group. Or reprocess the data frames in P2(1) if you have lost the unmerged data file. Now of course this will still give you a poor completeness if you have used a strategy to optimize data collection in the orthorhombic space group (you won't have collected enough data then for good completeness in the monoclinic space group). I hope this is clear ! HTH, Fred. On 24/03/13 11:20, Appu kumar wrote: I run the phenix.xtriage to evaluate the twining but it suggest no twining. When i reindex from C2221 to P21, the completeness of data reduced from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2 resolution. I do not understand why the completeness of data reduced so much on reindexing. please Can anyone explain this phenomenon. Thank you On 24 March 2013 13:30, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de wrote: the p21 c2221 ambivalence can mean severe twinning (i had a similar case just now - try several crystals from the same condition) ! What do the twinning statistics suggest? cheers, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/24/2013 7:46 AM, Appu kumar wrote: Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies (not just two copies but also one copy
[ccp4bb] molecular replacement problem.
Dear members, I am doing a molecular replacement of a transcription factor whose ligand binding structure(24000 Da) is available in PDB but not for the DNA binding(13000 Da). When i am searching for the two copies from ligand binding domain as a template model, i am getting very good solution but i am not getting any density for the DNA binding domain to build up in density. The space gorup is P 1 21 1 (4) and unit cell parameters are Unit Cell: 57.43 69.36 105.99 90.00 90.00 90.00. Please guide me how to get the complete model structure. Table below show the matthews statistics For estimated molecular weight 37000. Nmol/asym Matthews Coeff %solvent P(2.20) P(tot) _ 1 5.7178.46 0.00 0.01 2 2.8556.91 0.62 0.70 3 1.9035.37 0.37 0.29 4 1.4313.82 0.00 0.00 _ The phaser molecular replacement gives the following table. istogram of relative frequencies of VM values -- Frequency of most common VM value normalized to 1 VM values plotted in increments of 1/VM (0.02) --- relative frequency --- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ||||||||||| 10.00 - 8.33 - 7.14 - 6.25 - 5.56 - 5.00 - 4.55 - 4.17 - 3.85 -- 3.57 --- 3.33 -- 3.12 -- 2.94 (COMPOSITION*1) 2.78 --- 2.63 2.50 - 2.38 2.27 -- 2.17 --- 2.08 -- 2.00 -- 1.92 --- 1.85 --- 1.79 --- 1.72 - 1.67 - 1.61 - 1.56 - 1.52 - 1.47 * (COMPOSITION*2) 1.43 - 1.39 - 1.35 - 1.32 - 1.28 - 1.25 - $TABLE : Cell Content Analysis: $SCATTER :N*Composition vs Probability:0|3x0|1:1,2: $$ N*Composition Probability $$ loggraph $$ 1 0.306066 2 0.00141804 $$ Most probable VM for resolution = 2.27817 Most probable MW of protein in asu for resolution = 92664.2 Thank a lot in advance
[ccp4bb] molecular replacement for flexible domain of protein
Dear ccp4 user, I have problem in finding the phase for the flexible DNA binding domain of LTTR protein. The unit cell parameters are ,Unit Cell: 46.92 105.30 47.75 90.00 103.26 90.00, and space-Group P 1 21 1. Molecular weight of protein is 36000Da. When i am running phaser it showing that Most probable MW of protein in asu for resolution = 49714.8. Could you please guide me how to set the proper setting for finding the correct rotation function and translation function setting to get the phases for the full length protein. I m getting the phases for the ligand binding domain but not for the DNA binding domain. Could you please suggest me some tricks which would work to get the phase. Thanks a lot in advance Appu
[ccp4bb] simulated annealing omit map
Dear Users, I am refining a enzyme structure with ligand. I want to make the simulated annealing omit map of ligand including with 4 Angestron radius around the ligand. I am seeking your valuable advice to help me out. I know how to make omit map but i have not come accross making simmulated annealing omit map. I would appreciate your help. Thank you Appu
[ccp4bb] aligning structure and generating symetry mates
Dear all, Please guide me on right track in weired confusion. I have model structure(available in pdb) and a structure solved by molecular replacement as a dimer . Our structure does not giving correct tetramer by symmetry mates. Strangely when i am aligning our structure on model structure and generating symmetry mates of our structure to find the correct tetramer from dimer, i am getting the one which i want. Why the structure solved by us give tetramer on aligning with model structure. I do not know weather it is a right way to find the correct oligomeric state of protein or not. So please i reaquest you to suggest me with your valuable advice to overcome with this confusion. Thank you in advance Appu
[ccp4bb] Angle between monomer of a dimer
Dear all ccp4 users, I have solved the structure of a protein which is a dimer. On comparison with the other structure from PDB data bank, i found that structure solved by us show a tiltness in monomer-monomer arrangement of a dimer. May you please help me in findng the angle between two monomer. Do i need to calculate the centre of mass of the protein for calcualting the angle between them?. Thanks in advance please help me. Thank you Appu
Re: [ccp4bb] Angle between monomer of a dimer
Sir one more help plz i have the following matrix which one shall i use to calculate the angle. INFO:: coordinates transformed by orthonal matrix: | -0.6927, -0.7191,0.0564| | -0.4828,0.5204,0.7043| | -0.5358,0.4606, -0.7076| ( 76.04,-10.19, 28.64) Rotation - polar (omega,phi,kappa) 69.7525 112.368 160.045 Rotation - euler (alpha,beta,gamma) 85.4221 135.041 40.6865 Translation - Angstroms76.037 -10.1858 28.6373 If you won't mind please tell me how to calculathe angle from this. I am new to this type of problem. Thank you Appu On 24 August 2012 08:52, Appu kumar appu.kum...@gmail.com wrote: Thank you Sir for your kind eply. I will follow your instruction to calculate the angle. On 24 August 2012 00:38, Nikolina Sekulic nikolina.seku...@gmail.comwrote: Dear Appu, I had similar issue once. You can use Coot to overlay two structures and get the rotation matrix a1 a2 a3 b1 b2 b3 c1 c2 c3 from here you can calculate cosine of an angle using formula (a1+b2+c3-1)/2 Good luck, Nikolina Sekulic, PhD postdoctoral fellow University of Pennsylvania On Thu, Aug 23, 2012 at 2:30 PM, Appu kumar appu.kum...@gmail.comwrote: Dear all ccp4 users, I have solved the structure of a protein which is a dimer. On comparison with the other structure from PDB data bank, i found that structure solved by us show a tiltness in monomer-monomer arrangement of a dimer. May you please help me in findng the angle between two monomer. Do i need to calculate the centre of mass of the protein for calcualting the angle between them?. Thanks in advance please help me. Thank you Appu
Re: [ccp4bb] Mlphare problem
Parameter Shifts greater than 2.500
Sigma will be scaled by 0.500
Number of Phasing and Refinement Cycles = 30
Input Phasing Flags for each Cycle are
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
If phasing flag NE 0 - input phases will be combined with calculated
phases.
Phase Probabilities will be calculated every 10 degrees
Flag for Calculation of Atomic form factors in electrons = 1
Flag for printing of Correlation Matrices = 0
Flag for printing of Statistics on the Derivative Scale = 1
!--SUMMARY_END--/FONT/B
Reciprocal matrix Real matrix
0.01301 0.0 0.0 76.840.000.00
0.0 0.01202 0.00.00 83.210.00
0.0 0.0 0.010490.000.00 95.32
The orthogonalisation code is:
B PARALLEL TO KO C* PARALLEL TO LO
B*PARALLEL TO YO C PARALLEL TO ZO
The real matrix pre-multiplies column vector {x y z} in
fractional coordinates to get orthogonal coordinates. The
reciprocal matrix is its inverse. This is not the same as
PDB standard NCODE 1. These matrices are used when
refining anisotropic temperature factors, although the
orthogonal axis definition is irrelevant.
Data line--- LABOUT PHIB=PHIB FOM=FOM HLA=HLA HLB=HLB HLC=HLC HLD=HLD
MtzParseLabin: neither label recognised: HLA HLA
MtzParseLabin: neither label recognised: HLB HLB
MtzParseLabin: neither label recognised: HLC HLC
MtzParseLabin: neither label recognised: HLD HLD
MLPHARE: Error in label assignments in LABOUT
MLPHARE: Error in label assignments in LABOUT
Times: User: 0.1s System:0.0s Elapsed: 0:00
/pre
/html
!--SUMMARY_END--/FONT/B
it seems that it is having problem in label input
On 20 June 2012 16:16, Eleanor Dodson eleanor.dod...@york.ac.uk wrote:
You can View input command file, when working from the GUI.
Could you cut and paste this into your message - obviously some inmportant
parameter is not set, but I cant tell which without seeing that file.
Eleanor
On 20 June 2012 05:25, Appu kumar appu.kum...@gmail.com wrote:
Thank you for your reply,
I am running Malphare through CCP4 GUI interface. I am giving mtz file
and Se.pdb file as input and running it. I do not know running Malphare
through script. Please help me with heavy atom refinement in Malphare.
On 20 June 2012 03:49, Pete Meyer pame...@mcw.edu wrote:
It's not clear without looking at your input script, but it appears that
you've got a problem with either your LABIN, ATREF or ATOM lines. It might
be helpful to check out some of the example scripts distributed with ccp4.
Pete
Appu kumar wrote:
Dear CCP4 user,
I am facing problem in running Mlphare
for HEAVY ATOM REFINEMENT and PHASE CALCULATO. It is running successfully
but in output log file i am not getting any FOM for acentric. Log file
output of one of the cycle is
*** Finished refinement cycle 3
0 reflections were rejected for refinement
Figures of merit as functions of resolution shell
--**---
Resolution of each shell in angstroms:
19.88 11.818.406.525.334.503.903.44
Number of Measurements phased -ACENTRIC
TOTAL
20 107 280 498 721109213831342
5443
Mean Figure of Merit
0. 0. 0. 0. 0. 0. 0. 0.
0.
Number of Measurements phased -CENTRIC
TOTAL
29 62 98 120 139 161 158 145
912
Mean Figure of Merit
0. 0. 0. 0. 0. 0. 0. 0.
0.
Number of Measurements phased -ALL
TOTAL
49 169 378 618 860125315411487
6355
Mean Figure of Merit
0. 0. 0. 0. 0. 0. 0. 0.
0.
Compound 1 Se
-
Scale Factor1. Shift0.S. D.
0.
Overall B 0. Shift0.S. D.
0.000
--**-
Refinement Parameter = 0.00
Anomalous Refinement Parameter = 0.00
--**-
== CYCLE 4 ==**
=
One more thing Mlphare did not giving me anamolous shift for individual
Se site. Please guide me and suggest me your valuble ideas to overcome this
problem.
Thank you
Appu
[ccp4bb] Mlphare problem
Dear CCP4 user, I am facing problem in running Mlphare for *HEAVY ATOM REFINEMENT and PHASE CALCULATO. It is running successfully but in output log file i am not getting any FOM for acentric. Log file output of one of the cycle is ** * *** Finished refinement cycle 3 0 reflections were rejected for refinement Figures of merit as functions of resolution shell - Resolution of each shell in angstroms: 19.88 11.818.406.525.334.503.903.44 Number of Measurements phased -ACENTRIC TOTAL 20 107 280 498 7211092138313425443 Mean Figure of Merit 0. 0. 0. 0. 0. 0. 0. 0. 0. Number of Measurements phased -CENTRICTOTAL 29 62 98 120 139 161 158 145 912 Mean Figure of Merit 0. 0. 0. 0. 0. 0. 0. 0. 0. Number of Measurements phased -ALLTOTAL 49 169 378 618 8601253154114876355 Mean Figure of Merit 0. 0. 0. 0. 0. 0. 0. 0. 0. Compound 1 Se - Scale Factor1. Shift0.S. D.0. Overall B 0. Shift0.S. D. 0.000 --- Refinement Parameter = 0.00 Anomalous Refinement Parameter = 0.00 --- == CYCLE 4 === One more thing Mlphare did not giving me anamolous shift for individual Se site. Please guide me and suggest me your valuble ideas to overcome this problem. Thank you Appu
Re: [ccp4bb] Mlphare problem
Thank you for your reply, I am running Malphare through CCP4 GUI interface. I am giving mtz file and Se.pdb file as input and running it. I do not know running Malphare through script. Please help me with heavy atom refinement in Malphare. On 20 June 2012 03:49, Pete Meyer pame...@mcw.edu wrote: It's not clear without looking at your input script, but it appears that you've got a problem with either your LABIN, ATREF or ATOM lines. It might be helpful to check out some of the example scripts distributed with ccp4. Pete Appu kumar wrote: Dear CCP4 user, I am facing problem in running Mlphare for HEAVY ATOM REFINEMENT and PHASE CALCULATO. It is running successfully but in output log file i am not getting any FOM for acentric. Log file output of one of the cycle is *** Finished refinement cycle 3 0 reflections were rejected for refinement Figures of merit as functions of resolution shell --**--- Resolution of each shell in angstroms: 19.88 11.818.406.525.334.503.903.44 Number of Measurements phased -ACENTRIC TOTAL 20 107 280 498 721109213831342 5443 Mean Figure of Merit 0. 0. 0. 0. 0. 0. 0. 0. 0. Number of Measurements phased -CENTRIC TOTAL 29 62 98 120 139 161 158 145 912 Mean Figure of Merit 0. 0. 0. 0. 0. 0. 0. 0. 0. Number of Measurements phased -ALL TOTAL 49 169 378 618 860125315411487 6355 Mean Figure of Merit 0. 0. 0. 0. 0. 0. 0. 0. 0. Compound 1 Se - Scale Factor1. Shift0.S. D. 0. Overall B 0. Shift0.S. D. 0.000 --**- Refinement Parameter = 0.00 Anomalous Refinement Parameter = 0.00 --**- == CYCLE 4 ==** = One more thing Mlphare did not giving me anamolous shift for individual Se site. Please guide me and suggest me your valuble ideas to overcome this problem. Thank you Appu
[ccp4bb] pROBLEM IN RUNNING dm
Hello Dear all I am trying running dm (density modification) programe for density modification, but it giving error complaining about density map. It says, dm: (WFO) No density in map! Check input FOM present and nonzero. Please suggest me how to overcome this problem. I am density modification with SAD data , but dm giving error. Your help will be much appreciated. Thank you Appu
Re: [ccp4bb] pROBLEM IN RUNNING dm
thanks a lott sir It really helped me, Thank you for your kind and valuable information. Thank you Appu On 14 June 2012 20:53, James Holton jmhol...@lbl.gov wrote: This is a bug that arises when you import data with scalepack2mtz. The resulting mtz file is not sorted, and apparently this makes DM unhappy in a rather uninformative way. However, if you run the data through CAD before inputting it into DM, then it works just fine. That is, just use the CCP4 program called CAD to copy all the columns into a new *.mtz file. Like this: echo labin file 1 all | cad hklin1 crashes_dm.mtz hklout no_crash_dm.mtz By default, a do nothing CAD run like this updates the sorting information in the mtz header. I have no idea why scalepack2mtz does not sort the data, nor why DM cannot just fix it. Perhaps it lies in the nature of the CCP4 Suite where a particular task (like sorting) is supposed to be done by one program (such as SORTMTZ) and not many? If anyone is interested in reproducing this problem, I have an example input file and script here: http://bl831.als.lbl.gov/~**jamesh/pickup/for_kevin.tgzhttp://bl831.als.lbl.gov/%7Ejamesh/pickup/for_kevin.tgz To generate this input file, I used scalepack2mtz, TRUNCATE, and then MLPHARE. The test.com script runs DM. And yes, it did take me a while to figure this out. -James Holton MAD Scientist On 6/14/2012 4:58 AM, Appu kumar wrote: Hello Dear all I am trying running dm (density modification) programe for density modification, but it giving error complaining about density map. It says, dm: (WFO) No density in map! Check input FOM present and nonzero. Please suggest me how to overcome this problem. I am density modification with SAD data , but dm giving error. Your help will be much appreciated. Thank you Appu
Re: [ccp4bb] HKL2000 indexing problem
Hi peter,
I agreed with Kelly, You should try indexing your data in
different program. I recently collected a diffraction data which showed
problem in indexing in HKL2000, but data indexed very fine in imosflm
giving right cell parameter. So it is worthfull to index your data in
different program.
On 21 February 2012 21:31, Green, Todd gr...@cbse.uab.edu wrote:
**
This is a possibility, but probably you should be able to get the initial
index in this situation. The integration is a different story. To fix that
problem, usually you can change the def.site fix with: {alig,1} {180} as
opposed to 0.
-Todd
-Original Message-
From: CCP4 bulletin board on behalf of Jacob Keller
Sent: Tue 2/21/2012 9:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] HKL2000 indexing problem
Is the direction of rotation correct? I got some data that were going
the wrong way once.
JPK
This email was scanned with Mcafee's Anti-Virus appliance, but this
is no guarantee that no virus exists. You are asked to make sure you
have virus protection and that it is up to date.
[ccp4bb] RMSD of side chains
Dear ccp4 users, Would you please guide me how to calculate the RMSD of side chains alone without considering C-alpha backbone. Is/are there any program/programs availble which do this job. I want to know the RMSD of side chains for protein comparison. Thank you in advance. Appu
Re: [ccp4bb] RMSD of side chains
Firstly thanks to Robert Nicholls for making me aware of the software necessary for side chain RMSD calculation. I have installed and now going through manual to use it for exploiting the structural differences. Thanks a lot. Secondly, for Ethan Merritt, I am seeking the information for comparing the side chains RMSD for better comparison of structure. Please correct me if i am wrong, i want to elaborate more on what i am thinking. If we have refine the structure well so that issue of rotamers are fixed, then it is possible to take the advantage of side chain orientation for correctly understanding the trivial differences between homologous proteins and such differences harbouring good piece information for understanding protein structure-function relationship. Any kind of suggestion would be highly appreciated. Thank you Appu On 13 January 2012 21:53, Ethan Merritt merr...@u.washington.edu wrote: On Friday, 13 January 2012, Appu kumar wrote: Dear ccp4 users, Would you please guide me how to calculate the RMSD of side chains alone without considering C-alpha backbone. Is/are there any program/programs availble which do this job. I want to know the RMSD of side chains for protein comparison. What is the question that you are trying to answer? If you are going to disregard the mainchain position, then I would guess that you'd be better off comparing rotamer classes than comparing coordinates. Ethan Thank you in advance. Appu
[ccp4bb] RNA polymerase purification
Hello every one, Sorry for asking off topic question, I have purified different subunit of RNA polymerase, now i want to assemble it. Can you give me valuable suggestion regarding the gel filtration column and ion exchange column which will facilitate the purification of assembled RNA pol. I want to know name of gelfilteration column and other column which would be used to purify. I do not hav much knowledge regarding column so would you please help me by giving me your valuable suggestion. Your suggestion would be highly appreciated. Thank you.
