Re: [ccp4bb] Dose anyone see this ligand before?
hi Wei, This looks like MPD, 2-Methyl-2,4-pentanediol. May b u have added it as ur cryo-protectant. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” From: sonali dhindwal sonali11dhind...@yahoo.co.in To: ccp4...@hotmail.com ccp4...@hotmail.com Sent: Wednesday, 17 July 2013 9:44 AM Subject: Re: [ccp4bb] Dose anyone see this ligand before? hi Wei, This looks like MPD, 2-Methyl-2,4-pentanediol. May b u have added it as ur cryo-protectant. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” From: Wei Feng ccp4...@hotmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, 16 July 2013 9:05 PM Subject: [ccp4bb] Dose anyone see this ligand before? Dear all, I found some redundant density in my structure beween two molecule. (see picture 1 and 2) But I am not sure which ligand will be. Dose everyone see this ligand before? If so, can you tell the PDB code or send me the struture file? Thank you for your time! Wei
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
Dear All, Thanks for the reply. I will try process and refine the data again, will see if it improves. Thanks again to all. -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” From: Mark van Raaij mjvanra...@cnb.csic.es To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, 30 April 2013 1:42 AM Subject: Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit and 2VAK...average Bs for the twelve, sequence identical, chains vary from 28 to 53. On 29 Apr 2013, at 22:09, David Schuller wrote: On 04/29/13 12:26, Roger Rowlett wrote: FYI, I do know of one example of a solved structure where some of the molecules in the ASU are poorly defined. In 1EKJ, 4 of the 8 molecules in the ASU have low B-factors (mid 30s) and 4 have high B-factors (50s-60s). In the unit cell these layers alternate. It is possible, if everything else has been excluded, that your crystal has a similar crystal-packing issue. Another is 3HDH. Pig short chain L-3-hydroxyacyl-CoA dehydrongenase revisited: sequence analysis and crystal structure determination. Barycki, et al (1999) Protein Science 8: 2010-2018. Examination of the map in the region of subunit C revealed that the electron density was considerably weaker for the third subunit compared to the other subunits... -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
Dear Eleanor, Thanks for the suggestion, I just checked on the Zanuda program, it is also giving P1 as the best possible spacegroup for the molecule. and by not refining well, I meant for the electron density which is broken at many places at main chain, and poor electron density for the side chains which are otherwise good in other chains. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” From: Eleanor Dodson eleanor.dod...@york.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, 29 April 2013 4:36 PM Subject: Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit There isnt much information here! Funny that 3 chains are poor - does that mean one and a half heterodimers? I presume you have checked spacegroup? Zanuda will test to see if any higher symmetry is present.. Eleanor On 29 April 2013 06:02, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, We are working on a crystal structure with 12 molecules in an asymmetric unit. It is a heteromer and each chain is made up of two type of chains. Therefore there are 24 chains in an asymmetric unit. It is giving solution in P1. Rfactor and Rfree has reached 20.25 and 25.6 at a resolution of 1.8Angstrom All the chains are refining well and have good electron density except 3 chains which have very poor electron density in most of the region. Whereas, the crystal structure of variants of the same protein we have solved earlier refined well and has very good electron density in all the chains. What could be the possible reason for these three chains which are not refining properly. Any suggestions will be very useful. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.”
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
Dear All, Thanks for the help and suggestion Francis, we used molrep for the structure solution. As I told that we already have the structure of the same protein, but a variant. So, we used its single chain (heteromer) as a model for molecular replacement. and Eleanor, I deleted once that chain and did refinement, all I was getting was the small small broken electron density everywhere in that chain region. Thanks again. -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” From: Francis E. Reyes francis.re...@colorado.edu To: sonali dhindwal sonali11dhind...@yahoo.co.in Sent: Monday, 29 April 2013 10:03 PM Subject: Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit Where are the phases coming from? MR? F On Apr 28, 2013, at 10:02 PM, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, We are working on a crystal structure with 12 molecules in an asymmetric unit. It is a heteromer and each chain is made up of two type of chains. Therefore there are 24 chains in an asymmetric unit. It is giving solution in P1. Rfactor and Rfree has reached 20.25 and 25.6 at a resolution of 1.8Angstrom All the chains are refining well and have good electron density except 3 chains which have very poor electron density in most of the region. Whereas, the crystal structure of variants of the same protein we have solved earlier refined well and has very good electron density in all the chains. What could be the possible reason for these three chains which are not refining properly. Any suggestions will be very useful. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
[ccp4bb] Poor electron density in some of the chains in an asymmetric unit
Dear All, We are working on a crystal structure with 12 molecules in an asymmetric unit. It is a heteromer and each chain is made up of two type of chains. Therefore there are 24 chains in an asymmetric unit. It is giving solution in P1. Rfactor and Rfree has reached 20.25 and 25.6 at a resolution of 1.8Angstrom All the chains are refining well and have good electron density except 3 chains which have very poor electron density in most of the region. Whereas, the crystal structure of variants of the same protein we have solved earlier refined well and has very good electron density in all the chains. What could be the possible reason for these three chains which are not refining properly. Any suggestions will be very useful. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.”
[ccp4bb] Query regarding the use of anisotropic temperature factor and ideal rmsAngle and rmsBond length values
Dear All, We want little suggestion and knowledge regarding refinement of data in Refmac. We have a data with resolution upto 1.5A. Overall redundancy of 5.5 and 3.7 in high resolution bin. and I over Sigma is also 21 overall and 2.2 in last resolution bin. When we first did isotropic refinement we used automatic weighing term, which gave good Rfree and Rfactor of 18.4 and 16.9 but high rmsBond and rmsAngle of 0.027 and 2.5 respectively. We were able to improve rmsBond and rmsAngle values by decreasing weighing term to 0.5. But when we do anisotropic refinement with weighing term of 0.5 it gives Rfree, Rfactor and FOM of 16.8, 15.0 and 90.7 respectively. And rmsAngle and rmsBond of 0.0074 and 1.25. Now, we want to know what should be the ideal values for rmsAngle and rmsBond at such resolution. Secondly, if we can use anisotropic refinement with such data. All your suggestions will be highly valuable. Thanks in advance. -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.”
Re: [ccp4bb] Query regarding the use of anisotropic temperature factor and ideal rmsAngle and rmsBond length values
Dear All,
I want to thank all for your helpful and quick suggestions and insight.
It was a very good discussion and also helped in understanding the topic.
Thanks again.
--
Sonali Dhindwal
“Live as if you were to die tomorrow. Learn as if you were to live forever.”
--- On Mon, 18/3/13, Ian Tickle ianj...@gmail.com wrote:
From: Ian Tickle ianj...@gmail.com
Subject: Re: [ccp4bb] Query regarding the use of anisotropic temperature factor
and ideal rmsAngle and rmsBond length values
To: CCP4BB@JISCMAIL.AC.UK
Date: Monday, 18 March, 2013, 4:06 AM
On 17 March 2013 13:19, Robbie Joosten robbie_joos...@hotmail.com wrote:
Small
addition to Ian's comment. The value you give with 'weight auto $value'
is a starting value. Refmac will gradually change it if needed (it's
autoweighting after all) and your starting value
does matter somewhat. Based on Ian's advice PDB_REDO uses a starting
value of 2.50 which seems to do the trick most of the times.
Cheers,
Robbie
Good point, here's an example from a Refmac log file of that
happening in practice using Refmac_5.8.0031 (output redacted so not
showing every iteration):
Data line--- WEIGHT AUTO 0.95
Weight matrix 1.00564407E-02
Actual weight 0.9499 is applied to the X-ray term
Weight matrix 1.19658876E-02
Actual weight 0.9499 is applied to the X-ray term
Weight matrix 1.67442132E-02
Actual weight 1.234 is applied to the X-ray term
Weight matrix 2.25313306E-02
Actual weight 1.6054999 is applied to the X-ray term
Weight matrix 2.31216233E-02
Actual weight 1.6054999 is applied to the X-ray term
Weight matrix 2.32296251E-02
Actual weight 1.6054999 is applied to the X-ray term
Weight matrix 2.34125387E-02
Actual weight 1.6054999 is applied to the X-ray term
Weight matrix 3.04648653E-02
Actual weight 2.0871499 is applied to the X-ray term
Weight matrix 3.05170882E-02
Actual weight 2.0871499 is applied to the X-ray term
So here I inputted Wa = 0.95 (I deliberately chose it too small), and
Refmac calculates what the equivalent value of Wm (1.00564407E-02) would
have been (i.e. inputting WEIGHT MATRIX 1.00564407E-02 would have given
identical results to WEIGHT AUTO 0.95). However what is actually used
('Actual weight') in the refinement calculation is Wa. Then Wa is
automatically optimised according to Refmac's internal criteria, giving a
final optimised value Wa = 2.09. Note that Wm changes as the model is
improved even when Wa is kept fixed.
Cheers
-- Ian
Re: [ccp4bb] help regarding structure solution - high R values after MR
Dear All, Thanks a lot for all the suggestions and help. Dear Eleanor, I will check the mtz file as u mentioned for the spacegroup and Dear Pye, Thanks for the link, i will run it and will let you knw if i find any luck. Thanks again, Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Fri, 22/6/12, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: From: Eleanor Dodson eleanor.dod...@york.ac.uk Subject: Re: [ccp4bb] help regarding structure solution - high R values after MR To: CCP4BB@JISCMAIL.AC.UK Date: Friday, 22 June, 2012, 3:30 PM I wonder if this could have happened here? Some one in the lab has yet again been trapped by a feature?? of REFMAC. Say your MR solution is found to be in P21212 after you searched various orthorhombic SFs, but the input MTZ file has the space group still listed as P222 (i.e. the point group) then REFMAC will treat the solution as in P222, and NOT use the SG given on the PDB CRYST1 card.. You need to change the space group in the mtz header using one of the reflection utilities.CAD or SFTOOLS or REINDEX (without any reindexing..) all will rewrite the mtz file with the correct space group. It would be nice if REFMAC reported any discrepancy in SG assignment as a fatal error.. Eleanor On 22 Jun 2012, at 00:53, Valerie Pye wrote: Hi Sonali, You could try wide-search MR: https://portal.sbgrid.org/d/apps/wsmr/ Best of luck, val On 20 June 2012 19:13, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution
Dear Raaij, We have not done mass-spec on the band from SDS-PAGE to confirm if it is our desired protein or any other contaminant. So, cant say for sure. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Thu, 21/6/12, Mark J van Raaij mjvanra...@cnb.csic.es wrote: From: Mark J van Raaij mjvanra...@cnb.csic.es Subject: Re: [ccp4bb] help regarding structure solution To: sonali dhindwal sonali11dhind...@yahoo.co.in Date: Thursday, 21 June, 2012, 11:33 AM you didn't answer the most important question - are you 100% sure the protein in the crystal is not a contaminant? Unfortunately, these things happen... Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 21 Jun 2012, at 06:47, sonali dhindwal wrote: Dear All, Thanks a lot for your replies. Glad to found so much help. Clemens, cell parameters are, 38.0020 78.0240 56.3800 90. 102.2770 90., in P21 spacegroup. Raaij, Savvas, we have checked for the twining and no twining was detected. Nat, DEN is a good suggestion, i will definitely try it today. Roger, Molrep didn't fail but as i mentioned in the mail, it do give solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. But none of the solution fits in the electron density. And phaser didn't give any solution. We checked in pointless also, it was suggesting the same spacegroup. Matthew, Yes, we don't have the same crystal now. Garib, I will run the balbes server, and will let you know then. Robert, I tried using your server, and found few hits. Will run those templates for molecular replacement. Peter, We didnt had MBP tag in the protein. But your idea of doing limited proteolysis sounds good, and will definitely try that. Thanks again to all, for their kind and valuable help. I will write after trying all the things as suggested. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Thu, 21/6/12, Peter Hsu hsuu...@u.washington.edu wrote: From: Peter Hsu hsuu...@u.washington.edu Subject: Re: [ccp4bb] help regarding structure solution To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, 21 June, 2012, 5:08 AM Hi Sonali, Did you use MBP as your purification tag? That's around 45-50kDa if I remember right. If not, I've had a decent amount of luck using in situ proteolysis to get crystals of degraded fragments. Try a limited proteolysis first overnight at 4C at varying concentrations of trypsin, see which one gives a nice stable band after overnight. Use that same concentration to add to your protein stock before setting up drops and then try another screen. I always use freshly prepared trypsin stock instead of frozen solutions to make sure that the freeze thaw doesn't reduce activity of the trypsin and that batch to batch is reproducible. Best of luck, Peter
[ccp4bb] help regarding structure solution
Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure.Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali
Re: [ccp4bb] help regarding structure solution
Dear All, Thanks a lot for your replies. Glad to found so much help. Clemens, cell parameters are, 38.0020 78.0240 56.3800 90. 102.2770 90., in P21 spacegroup. Raaij, Savvas, we have checked for the twining and no twining was detected. Nat, DEN is a good suggestion, i will definitely try it today. Roger, Molrep didn't fail but as i mentioned in the mail, it do give solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. But none of the solution fits in the electron density. And phaser didn't give any solution. We checked in pointless also, it was suggesting the same spacegroup. Matthew, Yes, we don't have the same crystal now. Garib, I will run the balbes server, and will let you know then. Robert, I tried using your server, and found few hits. Will run those templates for molecular replacement. Peter, We didnt had MBP tag in the protein. But your idea of doing limited proteolysis sounds good, and will definitely try that. Thanks again to all, for their kind and valuable help. I will write after trying all the things as suggested. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Thu, 21/6/12, Peter Hsu hsuu...@u.washington.edu wrote: From: Peter Hsu hsuu...@u.washington.edu Subject: Re: [ccp4bb] help regarding structure solution To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, 21 June, 2012, 5:08 AM Hi Sonali, Did you use MBP as your purification tag? That's around 45-50kDa if I remember right. If not, I've had a decent amount of luck using in situ proteolysis to get crystals of degraded fragments. Try a limited proteolysis first overnight at 4C at varying concentrations of trypsin, see which one gives a nice stable band after overnight. Use that same concentration to add to your protein stock before setting up drops and then try another screen. I always use freshly prepared trypsin stock instead of frozen solutions to make sure that the freeze thaw doesn't reduce activity of the trypsin and that batch to batch is reproducible. Best of luck, Peter
[ccp4bb] Defining beamstop and error during indexing- moslfm
Dear All, We have collected a data for a protein crystal at SER-CAT Chicago and the detector is mar300.We are using mosflm to process the data.While indexing, the beamstop which it is taking is wrong, due to which it fails. I am trying to define the beamstop manually using tools like mask and spot search area. (it might be wrong) It will be highly appreciable if someone can please suggest if this the method we should use to define the beamstop or there is any site definition file which has to be used, as it is available for HKL2000 or how we have to define the beamstop in mosflm. -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.”
Re: [ccp4bb] Defining beamstop and error during indexing- moslfm
Dear Andrew and all the people for their help, I am providing mosflm the right beamstop and now, I am able to do the indexing, refinement and indexing too.Then I run scala for the output mtz file and it shows Rmerge too high 0.58and also when examining the spots and predictions in the image, it seems like it is not picking a lot of spots, so missing large number of reflections.So, any suggestions to correct it or am i doing something wrong. Thanks again for the help and suggestions -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Thu, 22/3/12, Andrew Leslie and...@mrc-lmb.cam.ac.uk wrote: From: Andrew Leslie and...@mrc-lmb.cam.ac.uk Subject: Re: [ccp4bb] Defining beamstop and error during indexing- moslfm To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, 22 March, 2012, 7:43 PM Dear Sonali, Just to add to Tim's reply, when you open the image with iMosflm, you can Drag and drop the direct beam position in the image display window. First, you have to click on the leftmost icon in the row of icons under the image filename (a green cross) which will display the direct beam position as read from the image header as a green cross on the image. You can then drag then over to where you think the beam position should be (judging from the position of the backstop shadow). If indexing still does not work, you can try doing a direct beam search from the indexing pane, which will do a grid search +/- 2mm from the current position. However, you need to beware of one thing. I believe that the rotation axis on the Sercat beamline goes in the opposite direction to most beam lines. In such cases, indexing using more than one image will never work, because the relative phi values will be wrong. You can try indexing with one image, and then if that works, try predicting the next image. If that prediction does not match, it is almost certainly because the rotation direction is wrong. To deal with this, choose the Settings-Experiment settings menu and click on the reverse phi box. Then you MUST repeat the spot search for each image being used in indexing, then it should all work. Best wishes, Andrew Dear All, We have collected a data for a protein crystal at SER-CAT Chicago and the detector is mar300.We are using mosflm to process the data.While indexing, the beamstop which it is taking is wrong, due to which it fails. I am trying to define the beamstop manually using tools like mask and spot search area. (it might be wrong) It will be highly appreciable if someone can please suggest if this the method we should use to define the beamstop or there is any site definition file which has to be used, as it is available for HKL2000 or how we have to define the beamstop in mosflm. -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.”
[ccp4bb] using pymol for making movie of change in protein structure
Dear All, My query is slightly out of scope of ccp4. I need some suggestions for making movie using pymol to show conformation of a reactive site loop, through the use of three crystal structure which are available showing this change. I have seen many people present the structural information in the form of movie, to show how the ligands come and the flexible residues moves giving change in the active site by movement of the flexible loops. I found emovie and installed the plugin but pymol installed on my system doesnt support the morph feature. Can someone please help in suggesting some other tool and tutorial for the same. Thanks in advance. Regards
Re: [ccp4bb] using pymol for making movie of change in protein structure
Dear All, Thanks a lot to all, for replying to my query.and yes Dr. Read, i m trying to make the movie for the first time but hopefully with the help of CCP4bb, i will be able to do the task. Thanks again.Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” --- On Wed, 21/3/12, Randy Read rj...@cam.ac.uk wrote: From: Randy Read rj...@cam.ac.uk Subject: Re: [ccp4bb] using pymol for making movie of change in protein structure To: CCP4BB@JISCMAIL.AC.UK Date: Wednesday, 21 March, 2012, 9:06 PM You've already had an answer about the morphing part of the question, so I'll just address the question about tools. In terms of making a movie, if you aren't already an expert in making movies with PyMol, I'd suggest starting first with ccp4mg. Once you have a set of coordinate files for the morph, it's very easy to make a nice movie. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 21 Mar 2012, at 14:52, sonali dhindwal wrote: Dear All, My query is slightly out of scope of ccp4. I need some suggestions for making movie using pymol to show conformation of a reactive site loop, through the use of three crystal structure which are available showing this change. I have seen many people present the structural information in the form of movie, to show how the ligands come and the flexible residues moves giving change in the active site by movement of the flexible loops. I found emovie and installed the plugin but pymol installed on my system doesnt support the morph feature. Can someone please help in suggesting some other tool and tutorial for the same. Thanks in advance. Regards
