[ccp4bb] residuewise rmsd for multiple chain superposition
Dear all, Is there any software which will print the RMSDs (residue wise, and not chain wise) for more-than-2 chains (having similar/same sequences) superposed together? Thanks in advance, sreetama
Re: [ccp4bb] residuewise rmsd for multiple chain superposition
Dear Sreetama, theseus can superpose multiple structures and reports RMSD per residue. http://www.theseus3d.org/ If your sequences are not identical and you want least squares superposition, run it like this: theseus_align -l -f *.pdb This will produce several output files. The file theseus_variances.txt contains the RMSD per residue. Cheers, Thomas On 02/21/2012 10:33 AM, sreetama das wrote: Dear all, Is there any software which will print the RMSDs (residue wise, and not chain wise) for more-than-2 chains (having similar/same sequences) superposed together? Thanks in advance, sreetama -- Thomas Holder MPI for Developmental Biology Spemannstr. 35 D-72076 Tübingen
[ccp4bb] proper or improper ncs?
Hi all This structure has the following ncs (output via phenix.simple_ncs_from_pdb) OPERATOR 1 CENTER: 18.3443 -55.4605 23.0986 ROTA 1:1.0.0. ROTA 2:0.1.0. ROTA 3:0.0.1. TRANS: 0.0.0. OPERATOR 2 CENTER: 37.0405 -23.8676 -14.9388 ROTA 1: -0.5444 -0.22020.8094 ROTA 2:0.8330 -0.02780.5526 ROTA 3: -0.09910.97510.1985 TRANS:45.3456 -78.7231 53.0085 It looks two-foldish but I'm not sure if it's proper or improper. (I'm trying to rationalize the lack of peaks on the self rotation maps). Any help would be appreciated. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
[ccp4bb] EMBO Course on Macromolecular Complexes, Grenoble 4-9 June 2012
Dear All, We are organizing an EMBO Practical Course on the Structural Characterization of Macromolecular Complexes. The course is primarily intended for Ph.D. students and postdocs working on challenging structural projects involving macromolecular complexes. WHEN: 4-9 June 2012 WHERE: Grenoble, France TOPICS INCLUDE: Expression purification of multi-subunit complexes Biophysical biochemical characterization of complexes Combining different structural methods (MX, NMR, SAXS, EM, MS) LECTURERS INCLUDE: Radu Aricescu (Oxford U.) Imre Berger (EMBL Grenoble) Elisabetta Boeri Erba (IBS Grenoble) Anne-Claude Gavin (EMBL Heidelberg) Kay Grünewald (Oxford U.) Edward Lemke (EMBL Heidelberg) Malene Ringkjobing (IBS Grenoble) Christophe Romier (IGBMC Illkirch) Vladimir Rybin (EMBL Heidelberg) Michael Sattler (Technische U. Munich) Helen Saibil (Birkbeck, U. London) Christiane Schaffitzel (EMBL Grenoble) Carsten Schultz (EMBL Heidelberg) Bertrand Séraphin (IGBMC Illkirch) Montserrat Soler López (IRB Barcelona) Dmitri Svergun (EMBL Hamburg) Song Tan (Pennsylvania State U.) SPONSORED BY: EMBO, P-CUBE, ESRF, Instruct APPLICATION DEADLINE: 4 April 2012 COURSE DETAILS REGISTRATION: http://events.embo.org/12-characterization/ Please forward this announcement to anyone who might be interested in attending this course. Best regards, Carlo Petosa, Darren Hart, Elspeth Gordon, Guy Schoehn, Winfried Weissenhorn Carlo Petosa Institut de Biologie Structurale (IBS) 41 rue Jules Horowitz, 38042 Grenoble, France Tel:+33 438 78 40 24 E-mail: carlo.pet...@ibs.fr
Re: [ccp4bb] proper or improper ncs?
Hi, I'm sure there's a way to do this in CCP4, but using some little jiffy programs I've collected over the years... That's a 133 degree rotation around an axis defined by the unit vector 0.290647, 0.624977, 0.724519, and there's a small screw component of about 2.4A along the axis, so it's an improper rotation. On the other hand, if you combine a crystallographic symmetry operator with that transformation, you might still find that it's equivalent to a proper rotation. Regards, Randy Read On 21 Feb 2012, at 13:47, Francis E Reyes wrote: Hi all This structure has the following ncs (output via phenix.simple_ncs_from_pdb) OPERATOR 1 CENTER: 18.3443 -55.4605 23.0986 ROTA 1:1.0.0. ROTA 2:0.1.0. ROTA 3:0.0.1. TRANS: 0.0.0. OPERATOR 2 CENTER: 37.0405 -23.8676 -14.9388 ROTA 1: -0.5444 -0.22020.8094 ROTA 2:0.8330 -0.02780.5526 ROTA 3: -0.09910.97510.1985 TRANS:45.3456 -78.7231 53.0085 It looks two-foldish but I'm not sure if it's proper or improper. (I'm trying to rationalize the lack of peaks on the self rotation maps). Any help would be appreciated. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder -- 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 RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] proper or improper ncs?
Hi Francis, a quick insertion of your rotation matrix into CONVROT (by Alexandre Urzhumtsev) gives the following equivalent polar rotation angles (definition as in X-PLOR/CNS or Rossmann, 1962): phi,psi,kappa : 111.86 128.68 133.38 291.86 51.32 226.62 For a proper two-fold, a kappa of 180 degrees would be expected. This looks (very) improper to me. Best regards, Dirk. Am 21.02.12 14:47, schrieb Francis E Reyes: Hi all This structure has the following ncs (output via phenix.simple_ncs_from_pdb) OPERATOR 1 CENTER: 18.3443 -55.4605 23.0986 ROTA 1:1.0.0. ROTA 2:0.1.0. ROTA 3:0.0.1. TRANS: 0.0.0. OPERATOR 2 CENTER: 37.0405 -23.8676 -14.9388 ROTA 1: -0.5444 -0.22020.8094 ROTA 2:0.8330 -0.02780.5526 ROTA 3: -0.09910.97510.1985 TRANS:45.3456 -78.7231 53.0085 It looks two-foldish but I'm not sure if it's proper or improper. (I'm trying to rationalize the lack of peaks on the self rotation maps). Any help would be appreciated. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder -- *** Dirk Kostrewa Gene Center Munich Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] proper or improper ncs?
Francis, It's very easy to spot a 2-fold rotation or screw because the matrix must be symmetric (or nearly so)**. Your matrix very obviously is not (i,e, A12 ne A21, A13 ne A31 etc). ** Proof: A rotation matrix is orthogonal, which implies inverse = transpose: A^-1 = A~. A 2-fold rotation is proper which implies AA = I or A^-1 = A. Take these together and you get A = A~ i.e. A is symmetric. Surprising how many people aren't aware of this! Cheers -- Iann On 21 February 2012 13:47, Francis E Reyes francis.re...@colorado.edu wrote: Hi all This structure has the following ncs (output via phenix.simple_ncs_from_pdb) OPERATOR 1 CENTER: 18.3443 -55.4605 23.0986 ROTA 1: 1. 0. 0. ROTA 2: 0. 1. 0. ROTA 3: 0. 0. 1. TRANS: 0. 0. 0. OPERATOR 2 CENTER: 37.0405 -23.8676 -14.9388 ROTA 1: -0.5444 -0.2202 0.8094 ROTA 2: 0.8330 -0.0278 0.5526 ROTA 3: -0.0991 0.9751 0.1985 TRANS: 45.3456 -78.7231 53.0085 It looks two-foldish but I'm not sure if it's proper or improper. (I'm trying to rationalize the lack of peaks on the self rotation maps). Any help would be appreciated. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] proper or improper ncs?
I've had a couple of requests for the jiffy program that interpreted the transformation, so here's the FORTRAN source code for decomp.f, for anyone who is interested. It's very simple, so all you should need to do is something like: gfortran decomp.f -o decomp to get an executable (or replace gfortran by g77 or f77, as appropriate). Put the matrix and vector into a file (say matvec.dat), then: decomp matvec.dat It takes a matrix and vector as four lines on standard input, then decomposes the transformation into a rotation around an axis, a point on the axis, and a translation parallel to the axis. Regards, Randy decomp.f Description: Binary data On 21 Feb 2012, at 14:01, Randy Read wrote: Hi, I'm sure there's a way to do this in CCP4, but using some little jiffy programs I've collected over the years... That's a 133 degree rotation around an axis defined by the unit vector 0.290647, 0.624977, 0.724519, and there's a small screw component of about 2.4A along the axis, so it's an improper rotation. On the other hand, if you combine a crystallographic symmetry operator with that transformation, you might still find that it's equivalent to a proper rotation. Regards, Randy Read On 21 Feb 2012, at 13:47, Francis E Reyes wrote: Hi all This structure has the following ncs (output via phenix.simple_ncs_from_pdb) OPERATOR 1 CENTER: 18.3443 -55.4605 23.0986 ROTA 1:1.0.0. ROTA 2:0.1.0. ROTA 3:0.0.1. TRANS: 0.0.0. OPERATOR 2 CENTER: 37.0405 -23.8676 -14.9388 ROTA 1: -0.5444 -0.22020.8094 ROTA 2:0.8330 -0.02780.5526 ROTA 3: -0.09910.97510.1985 TRANS:45.3456 -78.7231 53.0085 It looks two-foldish but I'm not sure if it's proper or improper. (I'm trying to rationalize the lack of peaks on the self rotation maps). Any help would be appreciated. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder -- 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 RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk -- 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 RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] HKL2000 indexing problem
Sounds exactly like something is off in your site.def file. I would look to the beam center. Also, have you tried indexing with another program? Mosflm perhaps. I find that if one program cannot index the data, another usually can (as long as the data isn't too horrible!). Thus I always try to process my data in several programs in parallel. Best, Kelly Daughtry *** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** On Mon, Feb 20, 2012 at 7:28 PM, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, I recently collected a data set off a single crystal and have had problems with indexing it. Every time I go pick peaks for indexing it constantly picks peaks that are just slightly off the actual peak. After indexing, it would always be that 2 of the 3 cell dimensions would be fairly normal, while the 3rd would have some impossible value such as 1. On some other occasions if it manages to pick peaks properly, and every time I go to index it, it gives back an error that I don't have enough peaks picked to index (picked nearly 500). I've tried using a number of different images to index from and have run into the same problem. Has anyone else run into these problems? Does anyone have any idea what might be wrong w/my dataset and/or crystal? Thanks in advance for any insight, Peter
Re: [ccp4bb] proper or improper ncs?
Hi, everybody, In addition to all comments, just as a pleasure to remind an old-fashion no computer way to calculate the rotation angle. The trace of a rotation matrix (the sum of its diagonal elements) is always equal to 2*cos(kappa)+1. Calculate it yourself for kappa = 180° and compare with the trace of the Francis's matrix. ROTA 1: -0.5444 -0.22020.8094 ROTA 2:0.8330 -0.02780.5526 ROTA 3: -0.09910.97510.1985 One can easily reject (still without a computer) other crystallographic angles. Best regards, Sasha Urzhumtsev -Message d'origine- De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de Ian Tickle Envoyé : mardi 21 février 2012 15:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] proper or improper ncs? Francis, It's very easy to spot a 2-fold rotation or screw because the matrix must be symmetric (or nearly so)**. Your matrix very obviously is not (i,e, A12 ne A21, A13 ne A31 etc). ** Proof: A rotation matrix is orthogonal, which implies inverse = transpose: A^-1 = A~. A 2-fold rotation is proper which implies AA = I or A^-1 = A. Take these together and you get A = A~ i.e. A is symmetric. Surprising how many people aren't aware of this! Cheers -- Iann On 21 February 2012 13:47, Francis E Reyes francis.re...@colorado.edu wrote: Hi all This structure has the following ncs (output via phenix.simple_ncs_from_pdb) OPERATOR 1 CENTER: 18.3443 -55.4605 23.0986 ROTA 1: 1. 0. 0. ROTA 2: 0. 1. 0. ROTA 3: 0. 0. 1. TRANS: 0. 0. 0. OPERATOR 2 CENTER: 37.0405 -23.8676 -14.9388 ROTA 1: -0.5444 -0.2202 0.8094 ROTA 2: 0.8330 -0.0278 0.5526 ROTA 3: -0.0991 0.9751 0.1985 TRANS: 45.3456 -78.7231 53.0085 It looks two-foldish but I'm not sure if it's proper or improper. (I'm trying to rationalize the lack of peaks on the self rotation maps). Any help would be appreciated. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] HKL2000 indexing problem
Hello Peter- Is it possible that you are shooting close to an axis? If you are shooting near parallel to one of the crystal axis, you will get values that are reasonable for 2 cell lengths but extremely low values for the third. Try indexing a frame that is say 30 degrees into the collection and see if you have the same problem. If the crystal is aligned with one axis along the direction of the x-ray beam, and the goniometer rotating around that direction then you may likely have to reorient the crystal and recollect the data. This may be why you it hasn't worked for several images. Outside of beam center, i think this is something for you to have a look at. -Todd -Original Message- From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Peter Hsu [hsuu...@u.washington.edu] Sent: Monday, February 20, 2012 6:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HKL2000 indexing problem Hi all, I recently collected a data set off a single crystal and have had problems with indexing it. Every time I go pick peaks for indexing it constantly picks peaks that are just slightly off the actual peak. After indexing, it would always be that 2 of the 3 cell dimensions would be fairly normal, while the 3rd would have some impossible value such as 1. On some other occasions if it manages to pick peaks properly, and every time I go to index it, it gives back an error that I don't have enough peaks picked to index (picked nearly 500). I've tried using a number of different images to index from and have run into the same problem. Has anyone else run into these problems? Does anyone have any idea what might be wrong w/my dataset and/or crystal? Thanks in advance for any insight, Peter 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.
Re: [ccp4bb] HKL2000 indexing problem
Is the direction of rotation correct? I got some data that were going the wrong way once. JPK
Re: [ccp4bb] HKL2000 indexing problem
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] Vacancy for a Data Analysis Scientist: Diffraction and crystallography
Dear All, Please see below details of a data analysis scientist post available within the scientific software team at Diamond. For full details please go to the web pages at: http://www.diamond.ac.uk/Home/Jobs/Current/DIA0686-TH.html Job Title: Data Analysis Scientist Job Reference: DIA0686/CG Post Type: Full time, permanent Division: Science Salary information: Circa £34k - a higher salary may be available for an exceptionally experienced and qualified candidate. Application deadline: 25/03/2012 Regards, Alun Ashton ___ Alun Ashton, alun.ash...@diamond.ac.uk Tel: +44 1235 778404 Scientific Software Team Leader, http://www.diamond.ac.uk/ Diamond Light Source, Chilton, Didcot, Oxon, OX11 0DE, U.K.
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] Modified Cys by TCEP?
Hi all, I recently encountered a modified surface cysteine residue in one of my structures. The protein is expressed in E.coli and my data is to 1.7Å so I am positive of the number of extra atoms. It really look like one of the tails (the Propanoic acid) of a TCEP molecule. TCEP was added during purification at 2 mM. I tried to look in pdb (or google) but I could not find any reference of that TCEP could do this. The modified Cystein is involved in a nice crystal contact (salt bridge) to a Lysine, and thereby stabilizing it further. Looking back at older structures of the same protein, I see the same modification of this Cys, but not as pronounced since they were done at lower resolution. Does anyone recognize this behavior of TCEP? Or, any other ideas? Björn Kauppi Structure and Design Karo Bio AB __ This e-mail may contain confidential information proprietary to Karo Bio AB and is meant for the intended addressee(s) only. Any unauthorized review, use, disclosure or distribution is prohibited. If you have received this message in error, please advise the sender and delete the e-mail and any attachments from your files. Thank you! __
[ccp4bb] Aggregated protein for crystallization
Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Aggregated protein for crystallization
Hey, it could be that you just have a big oligomer--any support for that in the relevant literature? A 10-mer would probably beat out an s200, no? Do you have any other way to ascertain the oligomeric state? Jacob On Tue, Feb 21, 2012 at 5:21 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Aggregated protein for crystallization
I probably it depends on whether you've got gunk or a functionally relevant oligomer in that void volume. Is it active? RecA and Rad51 both form open-ended head-to-tail oligomers and yet they still crystallize. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Tue, 21 Feb 2012 18:21:03 -0500 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Raji Edayathumangalam r...@brandeis.edu) Subject: [ccp4bb] Aggregated protein for crystallization To: CCP4BB@JISCMAIL.AC.UK Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Aggregated protein for crystallization
Hi, Your idea does sound really crazy but actually Jacob had made a very good valid point. Question is do you think your aggregate still functioning or not, if not, can you revive them in vitro and how effective is your refolding process if you are going to refold them? You may want to take a look at structures of some bacterial pore-forming proteins. Many of them can be expressed as large inclusion bodies in heterologous host of which we call aggregate. I know it sound fishy but after refolding and proper proteolytic activation, those proteins retain the ability to induce mortality to their host cell as good as the real one. Anyway, good luck with your endeavors! Puey On Tue, Feb 21, 2012 at 3:26 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Hey, it could be that you just have a big oligomer--any support for that in the relevant literature? A 10-mer would probably beat out an s200, no? Do you have any other way to ascertain the oligomeric state? Jacob On Tue, Feb 21, 2012 at 5:21 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Aggregated protein for crystallization
Well, depends on what 'aggregated' really means. If it implies reasonably weak oligomerization interaction - and it might not be too strong given that the oligomers remain soluble - a chaotropic crystallization agent (on the extreme end certain high salts, consult Hofmeister for chaotropicity) may rip such soluble aggregates apart or at least get them into a conformationally reasonably well defined state. Crystals do appear/transform even from precipitates on occasion. CD will tell you about the (secondary structure) folding state, not the aggregation state, DLS/MALS would give an estimate for and distribution of the aggregation state. With light scattering, you can also do some systematic experiments exploring what might reduce the aggregate size. I think soluble, defined secondary structure, and a lot of it, is already a good sign. BR On Tue, Feb 21, 2012 at 5:21 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] residuewise rmsd for multiple chain superposition
On Feb 21, 2012, at 4:00 PM, sreetama das wrote: Is there any software which will print the RMSDs (residue wise, and not chain wise) for more-than-2 chains (having similar/ same sequences) superposed together? In UCSF Chimera you can use the Match-Align tool to create a structure-based sequence alignment (or you can open an alignment file if you already have one). The the alignment will show per-residue RMSD as a bar chart across the top of the alignment (if you opened your own alignment file you'd have to use the Headers-RMSD menu entry to show the bar chart). You can save the numeric RMSD values to a file with Headers-Save. Also, Match-Align will report a couple of additional normalized RMSD scores, namely Structural Distance Measure and Q-score. Chimera home page: www.cgl.ucsf.edu/chimera Match-Align page: http://www.cgl.ucsf.edu/chimera/current/docs/ContributedSoftware/matchalign/matchalign.html sequence aignment viewer: http://www.cgl.ucsf.edu/chimera/current/docs/ContributedSoftware/multalignviewer/framemav.html --Eric Eric Pettersen http://www.cgl.ucsf.edu/home/pett And isn't sanity really just a one trick pony anyway? I mean all you get is one trick, rational thinking, but when you're good and crazy, oooh oooh oooh, the sky is the limit!-The Tick
Re: [ccp4bb] Putting Text into Movies
On Feb 21, 2012, at 4:00 PM, Jacob Keller wrote: is there a good way to put text labels into movies from pymol or otherwise? It would be helpful for conveying some ideas... As Jason pointed out, PyMOL has some useful facilities for this. Chimera has a tool for placing standalone text and arrows called 2D Labels. The arrows and text can be any color and the text can also be various sizes, styles (italics, etc.) and typefaces (serif etc.), all on a per-character basis. You can place text and arrows interactively with the mouse or via commands. Text can also be faded in or out as a movie plays/records. A pretty nice example use of 2D Labels in the DNA basics movie on this page: http://crc.nd.edu/index.php/cyberinfrastructure/visualization/80 Chimera home page: www.cgl.ucsf.edu/chimera 2D Labels page: http://www.cgl.ucsf.edu/chimera/current/docs/ContributedSoftware/2dlabels/2dlabels.html --Eric Eric Pettersen UCSF Computer Graphics Lab http://www.cgl.ucsf.edu
[ccp4bb] Available positions for biophysicists with strong experience in SPR
Colleagues: This is a bit off-topic, but there are two positions available in our group for scientists with experience in biophysics and SPR. The description of each position follows as does application instructions. The first position listed is for an experienced Ph.D.-level scientist; the second position for an experienced B.S./M.S.-level scientist. Novartis Institutes for BioMedical Research SPR / Solution Biophysics Scientist - Inv III / Sr Inv I, Structural Chemistry Requisition No. 93406BR NIBR - Novartis Institutes for BioMedical Research Emeryville, CA About Novartis Institute of Biomedical Research At Novartis Institutes for BioMedical Research (NIBR), the global research organization of Novartis, we are committed to discovering innovative medicines to cure disease and improve human health. By hiring the best academic, biotech, and pharmaceutical trained scientists, we have fostered an atmosphere for drug discovery where innovation is rewarded. It is ultimately the talent of the individual that determines our success, while our state-of-the-art technologies and resources enable these ideas to be realized. NIBR has sites in Cambridge, Massachusetts; Emeryville, CA; Basel, Switzerland; Horsham, UK; and Shanghai, China. Our Emeryville location focuses on early drug discovery efforts for the Oncology Disease Area and offers a variety of positions in Biology, Chemistry, and functions such as Technology, Patents, Research Sciences and other areas that support our scientific resources. About Structural Chemistry The NIBR Emeryville Structural Chemistry group is responsible for actively pursuing protein-ligand interaction studies using NMR spectroscopy, X-ray crystallography, and complementary biophysical methods to support drug discovery projects. The group also determines the structures of macromolecular drug targets either as apo-proteins or in complex with lead compounds to help guide structure-based drug design, and actively participates in the drug discovery process. We are seeking a multifaceted individual with a strong scientific background and relevant expertise in the use of surface plasmon resonance (SPR) and other biophysical methods, such as isothermal calorimetry, to discover small molecule inhibitors and activators of drug targets, particularly in the context of fragment-based lead discovery. Responsibilities: * Design, develop, and implement SPR and other biophysical assays suitable for hit identification and validation as part of ongoing fragment-based screening efforts. * Drive the implementation of new technological approaches and capabilities in regards to solution biophysics. * Create and implement project plans, and coordinate workflow and logistics to meet project goals and milestones. * Participate on multidisciplinary teams to evaluate new leads and to validate new targets. Qualifications: * Ph.D. in Biochemistry, Biophysics, or other relevant field, with 3-10 years of relevant postgraduate experience, including biotechnology and/or pharmaceutical industry experience. * In-depth knowledge of biophysics is required, and experience with SPR technology and its application in fragment-based screen are essential. Experience with biophysics assay technologies such as fluorescence-based techniques (FP, FRET), calorimetry (ITC, DSC), light scattering (StarGazer) are highly desirable. * Experience in structure-based drug design and the optimization of ligand-efficient fragments would be a plus. * Experience with laboratory instrumentation and automation, data analysis and management, and a sound knowledge of the drug discovery process is also highly desirable. * Some knowledge of current infectious disease and oncology research is a plus. * Ability to devise experimental strategies and execute them in a timely manner to produce goal-oriented results is essential. * Ability to work with Novartis scientists from multiple disciplines is required and exceptional written, verbal, and team skills are essential. * Demonstrated solid track record of scientific achievements is required. To apply for this position, please visit www.novartis.comhttp://www.novartis.com, click on the Careers link, pull the menu down to Job Search, and search for job ID 93406BR. Solution Biophysics Research Associate - Scientist II Requisition No. 93761BR NIBR - Novartis Institutes for BioMedical Research Emeryville, CA Responsibilities: * Working with PhD-level scientists to develop and implement SPR and other biophysical assays suitable for hit identification and validation as part of ongoing fragment-based screening efforts. * Implement project plans, and coordinate workflow and logistics to meet project goals and milestones. * Participate on multidisciplinary teams to evaluate new leads and to validate
Re: [ccp4bb] Aggregated protein for crystallization
Hi, Did you try using a different column like Superose 6? This column works well to separate large molecular weight proteins including oligomers. Ideally if your solution is not cloudy (coming out of void volume) those are not aggregates those might be oligomers. HTH, Shya On Tue, Feb 21, 2012 at 6:21 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Server or software for B factor analysis
Dear All, Will you please tell me a server of software which can draw a curve for the B factor of the atoms in a protein PDB file from the first residue to the residue?Or a server or software by which we can easily order the B factors of the atoms in the PDB file according to the B factor in decrease or in increase? Or to get the residues with the highest B factor and the lowest B factor? Cheers, Dialing
Re: [ccp4bb] Server or software for B factor analysis
Hi Dialing, Will you please tell me a server of software which can draw a curve for the B factor of the atoms in a protein PDB file from the first residue to the residue?Or a server or software by which we can easily order the B factors of the atoms in the PDB file according to the B factor in decrease or in increase? Or to get the residues with the highest B factor and the lowest B factor? I know one, you can do it in Excel (it is not Phenix !). Hope this helps, Pavel
[ccp4bb] R-mergd-F
Hi all , I will like to know which program we can use to calculate R-mergd-F( not Rmerge) between two data sets, or more generally R factor between two data sets. Thanks in advance, Regards, ARKO -- *ARKA CHAKRABORTY* *CAS in Crystallography and Biophysics* *University of Madras* *Chennai,India*
Re: [ccp4bb] Best method for weighted averaging of Friedel pairs?
Hi all, Can someone put in links of a few articles relevant to the above discussion? ie where this kind of strategy was helpful in a specific practical situation? Thanks in advance, Regards, ARKO On Tue, Feb 21, 2012 at 4:46 AM, ccp4 c...@ysbl.york.ac.uk wrote: This is a case where it is really helpful to keep some record of the unmerged integrated data. And again rejecting the odd outlier does no harm to most analyses.. I like to use scala to check for outliers looking at all i+ I- measurements; if there is a wild discrepancy for weak anomalous signals you have probably found an outlier which is best rejected. If you have a huge anomalous signal with good redundancy you probably shouldnt use Imean and the anomalous difference will be I= -I- with SD = sqrt (VarI+ VarI-). Most software which uses that signal will check for outliers in the Anom difference lists too, and it is usually safest to exclude them from anom site searches, and phase calculations. Eleanor On 14.02.2012 06:30, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Markus, why don't you reintegrate the data with hkl2000 telling the program to treat them as non-anomalous data-set? This should give you scalepack output with the Bijvoet pairs merged and overcome the problem you describe. Cheers, Tim On 02/13/2012 06:19 PM, Markus Meier wrote: On 11/02/12 02:52 PM, Bryan Lepore wrote: did you ever get a response on this? it is interesting but nobody posted publicly. -Bryan Dear Bryan, so far no one replied ... so please find my answer below. If someone disagrees, please post. None of the methods I have described are appropriate. If the negative Bijvoet mates and the positive Bijvoet mates have been merged separately to one intensity value for each (i.e. I+ or I-) plus the associated standard deviation (sigI+ or sigI-), any weighted method to calculate the mean will bias the intensity to either the I+ or the I-. Therefore the only appropriate method is to use the unweighted mean: Imean = 0.5*( I+ + I- ) sigImean = 0.5 * sqrt( sigI+^2 + sigI-^2 ) The only CCP4 program I found that actually does this is mtzMADmod. This method also has the advantage that the original intensity values of I+ and I- can be reconstructed from the mean and the anomalous difference (albeit with the loss of the original standard deviations). Method 1 (scalepack2mtz) should not be used. The resulting value is not the best estimate (maximum likelihood) Method 2 (in book by B. Rupp) gives the maximum likelihood average in case that the reflections are equivalent and is thus appropriate for the merging of the negative (or positive) set of Bijvoet mates, centric reflections (where the anomalous differences are zero) or in the case of an non-anomalous dataset the merging of symmetry equivalent reflections. Method 3 gives a more realistic sigma value in the case that the individual intensity values are far apart and their individual standard deviations are small. Consider the example I have posted: I+: 23841.50 sigI+: 634.01 I-: 9628.57, sigI-: 264.75 Method 2: Imean=11738.95, sigIMean=244.31 Method 3: Imean=11738.95, sigIMean=7106.47 If the I+ and I- values above actually were symmetry equivalent reflections in an non-anomalous dataset, the sigImean from method 2 is ridiculously small and method 3 gives a far more realistic value. If method 3 is the best mathematical solution to this problem I am not able to judge and I have to trust the statistician (or programmer) who implemented this solution. Cheers, Markus On 10/02/12 01:47 PM, Markus Meier wrote: Dear all, I have a anomalous dataset, processed in HKL2000. Scalepack outputs a file containing the separately merged sets of the Friedel pairs I- and I+ and their standard deviations sigI+ and sigI-. Scalepack does not output the averaged intensities (Imean) and the standard deviations (sigIMean). The CCP4 program truncate that I use to convert the intensities to amplitudes requires Imean, I- and I+ and the respective standard deviations in its input file. I have now found at least three different methods to generate the averaged intensities from the Friedel pairs: 1) scalepack2mtz uses standard deviations for the weights: weights w = 1/sigI Imean = (w+*I+ + w-*I- ) / (w+ + w-) sigImean = 1 / (w+ + w-) 2) Method described in Biomolecular crystallography by Bernhard Rupp, p. 332/333 to average symmetry equivalent reflections uses variances for the weights: weight w = 1/sigI^2 Imean = (w+*I+ + w-*I- ) / (w+ + w-) sigImean = 1 / sqrt(w+ + w-) 3) Method used in cctbx function miller.set.average_bijvoet_**mates() that calls generic merge.merge_equivalent_obs(): same as methods 2, except that sigImean is the larger of either a) sigImean = 1 / sqrt(w+ + w-) or b) sigImean = sqrt( wvariance ) where wvariance = (w+ + w-) / [ (w+ +
[ccp4bb] Evidence for hydrogen bonds between adjacent residues
Hi all, This is a general question regarding the formation of hydrogen bonds in proteins. I have found hydrogen bond between the back-bone atoms of the adjacent residues using computational methods, ie, between NH4-CO5 of enkephalins. Is this kind of feature acceptable and is there any such experimental evidence? I could'nt find any reference. Thanks in advance, Regards, ARKO -- *ARKA CHAKRABORTY* *CAS in Crystallography and Biophysics* *University of Madras* *Chennai,India*
[ccp4bb] effects of salt on twinned crystals
Hi all, Thanks to all those who responded to my query about indexing my dataset. I'm beginning to suspect that the crystals are twinned. the crystals initially started as fused plates which were then optimized to single crystals that diffracted poorly (3.5-4). I then set up an additive screen, found an additive that changed crystal form (looked more like the plates from earlier), went through much optimization and got what appeared to be single rods, that diffracted much more strongly (2.5-3), although some crystals in the same drop looked like fused crystals, despite having single rods. The initial condition used LiCl as the salt, which during the optimization I changed to NaCl. I'm now looking for ideas to see how much more I can optimize this condition since I haven't found crystals in any other conditions (even did a seed screen). Does anyone have any experience with varying the different salts to get untwinned crystals? KCl better than NaCl? Ammonium chloride better than the others? Been looking at the Hofmeister series to get ideas. Long post I realize, but many thanks for any previous experiences with salts. Peter
