[ccp4bb] Biological Structures Group BCA Winter Meeting, Reading, UK - Wed 15th Dec - FINAL PROGRAMME -
Dear All, We are pleased to provide the final programme for the BSG Winter Meeting. ***Student bursaries are now available*** To be eligible, student participants are asked to submit an abstract (max 200 words) for a poster presentation and provide a letter of recommendation from their supervisor. We look forward to welcoming you to Reading! Yours, Kim BSG BCA Winter Meeting 2010 schedule.doc Description: BSG BCA Winter Meeting 2010 schedule.doc
Re: [ccp4bb] Can LSQKAB calculate RMSTAB for two pre-superposed structures?
Yes - I think there is a button on the GUI to click? Eleanor On 11/24/2010 01:58 AM, Huiying Li wrote: Another question on RMSD: I have two structures of the same protein superposed with the LSQ Superpose in Coot by matching the first ~100 residues of the N-terminal domain. Now I'd like to calculate the pair-wise RMSD for the entire pre-superposed structures (~400 residues). Can LSQKAB output RMSTAB for the two input PDB files without doing its own FIT/MATCH calculation? Thanks, Huiying ___ Huiying Li, Ph. D Department of Molecular Biology and Biochemistry Natural Sciences I, Rm 2443 University of California at Irvine Irvine, CA 92697, USA Tel: 949-824-4322(or -1953); Fax: 949-824-3280 email: h...@uci.edu
Re: [ccp4bb] Strange density on Serine oxygen.
Are you sure the sequence is right? It looks like tyrosine Phil On 24 Nov 2010, at 12:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] Strange density on Serine oxygen.
look like tyrosines to me! Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.researcherid.com/rid/B-3678-2009 On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] Strange density on Serine oxygen.
Hi Vinson Beyond the possibility for another type of residue as already suggested by Phil and Mark, there is also the possibility of O-linked glycosylation of the serine and threonine, if your protein undergoes such post-translational modification and it has been expressed via an expression system that processes the protein in that way. Ser/Thr tandems are well known targets for O-glycosylation (http://www.cbs.dtu.dk/databases/OGLYCBASE/). best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] Strange density on Serine oxygen.
Dear Savas and all, Thank you very much for all your quick suggestions. I have tried Tyr and it turns out to fit the density very well. I will have the protein sequenced again to see if it is wrong sequece or O-linked glycosylation. I'll let you know if it turns out to be O-linked glycosylation. All the best, Vinson 发件人： Savvas Savvides savvas.savvi...@ugent.be 收件人： Vinson LIANG lwg_conrad_1...@yahoo.com.cn 抄 送： CCP4BB@JISCMAIL.AC.UK 发送日期： 2010/11/24 (周三) 9:27:31 下午 主 题： Re: [ccp4bb] Strange density on Serine oxygen. Hi Vinson Beyond the possibility for another type of residue as already suggested by Phil and Mark, there is also the possibility of O-linked glycosylation of the serine and threonine, if your protein undergoes such post-translational modification and it has been expressed via an expression system that processes the protein in that way. Ser/Thr tandems are well known targets for O-glycosylation (http://www.cbs.dtu.dk/databases/OGLYCBASE/). best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] Strange density on Serine oxygen.
Hi Vinson, along these lines: did you check the molecular weight of your protein with MS? This should help to answer if the molecular weight deviates from the expected one. Best wishes, Linda Savvas Savvides schrieb: Hi Vinson Beyond the possibility for another type of residue as already suggested by Phil and Mark, there is also the possibility of O-linked glycosylation of the serine and threonine, if your protein undergoes such post-translational modification and it has been expressed via an expression system that processes the protein in that way. Ser/Thr tandems are well known targets for O-glycosylation (http://www.cbs.dtu.dk/databases/OGLYCBASE/). best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] Strange density on Serine oxygen.
Hi Vinson is O-glycosylation possible at all given the origin of the protein and the expression system used? best Savvas On 24 Nov 2010, at 14:42, Vinson LIANG wrote: Dear Savas and all, Thank you very much for all your quick suggestions. I have tried Tyr and it turns out to fit the density very well. I will have the protein sequenced again to see if it is wrong sequece or O-linked glycosylation. I'll let you know if it turns out to be O-linked glycosylation. All the best, Vinson 发件人： Savvas Savvides savvas.savvi...@ugent.be 收件人： Vinson LIANG lwg_conrad_1...@yahoo.com.cn 抄 送： CCP4BB@JISCMAIL.AC.UK 发送日期： 2010/11/24 (周三) 9:27:31 下午 主 题： Re: [ccp4bb] Strange density on Serine oxygen. Hi Vinson Beyond the possibility for another type of residue as already suggested by Phil and Mark, there is also the possibility of O-linked glycosylation of the serine and threonine, if your protein undergoes such post-translational modification and it has been expressed via an expression system that processes the protein in that way. Ser/Thr tandems are well known targets for O-glycosylation (http://www.cbs.dtu.dk/databases/OGLYCBASE/). best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] Strange density on Serine oxygen.
Phosphoserine ? शेखर चिं मांडे हैदराबाद On Wed, Nov 24, 2010 at 5:40 PM, Vinson LIANG lwg_conrad_1...@yahoo.com.cnwrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang --
Re: [ccp4bb] Strange density on Serine oxygen.
This density does not look at all like O-glycosylation. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Savvas Savvides Sent: Wednesday, November 24, 2010 2:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density on Serine oxygen. Hi Vinson is O-glycosylation possible at all given the origin of the protein and the expression system used? best Savvas On 24 Nov 2010, at 14:42, Vinson LIANG wrote: Dear Savas and all, Thank you very much for all your quick suggestions. I have tried Tyr and it turns out to fit the density very well. I will have the protein sequenced again to see if it is wrong sequece or O-linked glycosylation. I'll let you know if it turns out to be O-linked glycosylation. All the best, Vinson 发件人： Savvas Savvides savvas.savvi...@ugent.be 收件人： Vinson LIANG lwg_conrad_1...@yahoo.com.cn 抄 送： CCP4BB@JISCMAIL.AC.UK 发送日期： 2010/11/24 (周三) 9:27:31 下午 主 题： Re: [ccp4bb] Strange density on Serine oxygen. Hi Vinson Beyond the possibility for another type of residue as already suggested by Phil and Mark, there is also the possibility of O-linked glycosylation of the serine and threonine, if your protein undergoes such post-translational modification and it has been expressed via an expression system that processes the protein in that way. Ser/Thr tandems are well known targets for O-glycosylation (http://www.cbs.dtu.dk/databases/OGLYCBASE/). best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] unusual diffraction spots
I think there may be two effects going on here: I think the ears on the round spots which also feature on the more rod shaped spots if you look closely could be related to a misalignment of the beamline optics. I think the change in spot shape from round to rod shaped is due to the crystal quality. Do the ears only feature on this image of this crystal or do they appear on other images? If the ear effect is a one off then that would tend to suggest it isn't a beamline optic effect. Liz Dr. Liz Duke Principal Beamline Scientist Diamond Light Source Harwell Science and Innovation Campus Chilton OX11 0DE UK Tel. 01235 778057 Mob. 07920 138148 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Hubing Lou Sent: 24 November 2010 14:09 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unusual diffraction spots Dear CCP4BBer, I recently collected a dataset at synchrotron. The diffraction was quite anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What unusual is in the diffraction image (see the attached file), clearly at low resolution there were some spots with tails (two ears) and at the high resolution shell the spots turned to be rod-shaped. Please, can anyone explain how this could be? Is this related to the anisotropy? The protein was N-terminal his6-tagged, we are currently preparing new samples with the His-tag removed. But any other suggestions are also very welcomed. Regards, Hubing
Re: [ccp4bb] unusual diffraction spots
Hi, The ears extended to other images but not all of them, some images do not show any signs like this. Thanks for replying, Liz. Best, Hubing On Wed, Nov 24, 2010 at 10:26 PM, elizabeth.d...@diamond.ac.uk wrote: I think there may be two effects going on here: I think the “ears” on the round spots which also feature on the more rod shaped spots if you look closely could be related to a misalignment of the beamline optics. I think the change in spot shape from round to rod shaped is due to the crystal quality. Do the “ears” only feature on this image of this crystal or do they appear on other images? If the ear effect is a one off then that would tend to suggest it isn’t a beamline optic effect. Liz Dr. Liz Duke Principal Beamline Scientist Diamond Light Source Harwell Science and Innovation Campus Chilton OX11 0DE UK Tel. 01235 778057 Mob. 07920 138148 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Hubing Lou *Sent:* 24 November 2010 14:09 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] unusual diffraction spots Dear CCP4BBer, I recently collected a dataset at synchrotron. The diffraction was quite anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What unusual is in the diffraction image (see the attached file), clearly at low resolution there were some spots with tails (two ears) and at the high resolution shell the spots turned to be rod-shaped. Please, can anyone explain how this could be? Is this related to the anisotropy? The protein was N-terminal his6-tagged, we are currently preparing new samples with the His-tag removed. But any other suggestions are also very welcomed. Regards, Hubing -- 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] Strange density on Serine oxygen.
Hi Savvas, You're right. It shouldn't be O-glycosylation, since the protein is from Archaea and expressed in E. coli. Most possiblly, I think it's something wrong with the sequence. Thanks for your help, Best, Vinson 发件人： Savvas Savvides savvas.savvi...@ugent.be 收件人： Vinson LIANG lwg_conrad_1...@yahoo.com.cn 抄 送： CCP4BB@JISCMAIL.AC.UK 发送日期： 2010/11/24 (周三) 9:48:28 下午 主 题： Re: [ccp4bb] Strange density on Serine oxygen. Hi Vinson is O-glycosylation possible at all given the origin of the protein and the expression system used? best Savvas On 24 Nov 2010, at 14:42, Vinson LIANG wrote: Dear Savas and all, Thank you very much for all your quick suggestions. I have tried Tyr and it turns out to fit the density very well. I will have the protein sequenced again to see if it is wrong sequece or O-linked glycosylation. I'll let you know if it turns out to be O-linked glycosylation. All the best, Vinson 发件人： Savvas Savvides savvas.savvi...@ugent.be 收件人： Vinson LIANG lwg_conrad_1...@yahoo.com.cn 抄 送： ccp...@jiscmail.ac.uk 发送日期： 2010/11/24 (周三) 9:27:31 下午 主 题： Re: [ccp4bb] Strange density on Serine oxygen. Hi Vinson Beyond the possibility for another type of residue as already suggested by Phil and Mark, there is also the possibility of O-linked glycosylation of the serine and threonine, if your protein undergoes such post-translational modification and it has been expressed via an expression system that processes the protein in that way. Ser/Thr tandems are well known targets for O-glycosylation (http://www.cbs.dtu.dk/databases/OGLYCBASE/). best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] Strange density on Serine oxygen.
Hi Linda and all, Thank you very much for your suggestion. I just help to solve the structure of this protein. And I'm not sure if the protein is properly sequenced first. I'll confirm the sequence first. I'll let you know if it is not the sequence's problem. I very appreciate help from all you guys, so quick and helpful. Best wishes, Vinson 发件人： Linda Schuldt lschu...@mb.au.dk 收件人： CCP4BB@JISCMAIL.AC.UK 发送日期： 2010/11/24 (周三) 9:35:33 下午 主 题： Re: [ccp4bb] Strange density on Serine oxygen. Hi Vinson, along these lines: did you check the molecular weight of your protein with MS? This should help to answer if the molecular weight deviates from the expected one. Best wishes, Linda Savvas Savvides schrieb: Hi Vinson Beyond the possibility for another type of residue as already suggested by Phil and Mark, there is also the possibility of O-linked glycosylation of the serine and threonine, if your protein undergoes such post-translational modification and it has been expressed via an expression system that processes the protein in that way. Ser/Thr tandems are well known targets for O-glycosylation (http://www.cbs.dtu.dk/databases/OGLYCBASE/). best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
Re: [ccp4bb] unusual diffraction spots
It seems that the Fourier cat is up to no good again.. BR From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Hubing Lou Sent: Wednesday, November 24, 2010 6:09 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unusual diffraction spots Dear CCP4BBer, I recently collected a dataset at synchrotron. The diffraction was quite anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What unusual is in the diffraction image (see the attached file), clearly at low resolution there were some spots with tails (two ears) and at the high resolution shell the spots turned to be rod-shaped. Please, can anyone explain how this could be? Is this related to the anisotropy? The protein was N-terminal his6-tagged, we are currently preparing new samples with the His-tag removed. But any other suggestions are also very welcomed. Regards, Hubing
[ccp4bb] Shape complementarity for nucleic acids
Hi! I am trying to calculate shape complementarity using SC in CCP4 for nucleic acids. SC stopped with an error no radius found for atoms of nucleic acid residues. I can edit in the radii for those atoms in sc_radii.lib, but am wondering what would the recommended radii be, especially for phosphorus? Thanks. -Sam
[ccp4bb] Protein sequencing service?
Can anyone please recommend a UK protein sequencing service. Our protein is dimeric with reported molecuar weight of about 85kDa.Our funds are somewhat limited. Thanks in advance. Rex Palmer Birkbeck College, London RexPalmer2010.homestead.com
Re: [ccp4bb] Protein sequencing service?
Hey Rex, Jeff Keen at Leeds has sequenced some of our proteins with good results. http://www.fbs.leeds.ac.uk/proteomics Andreas On 24/09/2010 4:46, Rex Palmer wrote: Can anyone please recommend a UK protein sequencing service. Our protein is dimeric with reported molecuar weight of about 85kDa.Our funds are somewhat limited. Thanks in advance. Rex Palmer Birkbeck College, London RexPalmer2010.homestead.com -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
[ccp4bb] Seeking used single crystal diffraction components - Detectors, Cryo-genic systems, X-rays sources
To All, Seeking used single crystal diffraction components - Detectors, Cryo-genic systems, X-rays sources. We are setting up several diffraction labs and are currently seeking system components. Any availability - donation, for sale , salvage. Thanks. Peace, Archie http://adicllc.com http://adicllc.com image001.gif
Re: [ccp4bb] Problem with finding of spots
Dear colleagues, I would like to express my thanks for all of your responses. I was able to find some reasonable initial space group hits, and now I have to fight against divergent refinement of cell parameters, and other things, but I hope I can solve this difficulties in some time. Actually, I have tried more than half of your suggestions before, but I still learned a lot from you. I think that this community is so far the best what internet offers. ;) Many thanks again. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz 2010/11/23 Petr Kolenko kole...@imc.cas.cz Dear colleagues, I am working on one dataset that is hard to process. The data are about 3A of resolution. As we are not able to reproduce the experiment again, I have to use this one, collected in a dirty way. The problem starts immediately with finding of spots. I have tried HKL2000, XDS, D*trek, ipmosflm, imosflm, but none of them gave a good read-out of the images. All the programs find some spots in wrong positions and the real spots are not covered. Here is an example: http://kolda.webz.cz/image-predictions.jpg The data were collected in-house, Saturn 944++ CCD, and all the necessary information should be in the header properly. I checked the distance, other parameters, but the problem is with finding of correct or real spots on the image. This should be even header-independent, should not? All the programs fail (or even crash) in this routine. Does anyone have any suggestion, please? Btw, we have several structures in the PDB from this experimental setup. This is the first problem I have met. Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
[ccp4bb] Postdoctoral position at Karolinska Institutet
Our group has an opening for a highly motivated postdoctoral fellow, to study the structure of mammalian ZP domain-containing proteins that mediate egg-sperm interaction at the beginning of fertilization. Parallel projects on different ZP domain proteins are also available. For further information, see Monné et al. Nature 456:653-657 (4 December 2008) [http://www.ncbi.nlm.nih.gov/pubmed/19052627] and Han et al. Cell 143:404-415 (29 October 2010) [http://www.ncbi.nlm.nih.gov/pubmed/20970175], as well as our web site [http://jovinelab.org]. The candidate must have significant experience in all aspects of macromolecular crystallography, from DNA cloning to structure refinement. Previous experience with protein expression using mammalian and/or insect cells, as well as familiarity with extracellular matrix proteins, will be an advantage. A good publication record in international journals, fluency in english and a strong personal drive to excel in science are required. To apply for this position, please send your full application including motivation letter, CV and the names and addresses of three referees to Luca Jovine (luca.jov...@ki.se). Luca Jovine, Ph.D. Group Leader EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 57 Huddinge, Sweden Voice: +46.(0)8.6083-301 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org
Re: [ccp4bb] Can LSQKAB calculate RMSTAB for two pre-superposed structures?
Thanks for your reply, Eleanor. The problem I had with LSQKAB is that it ONLY outputs the RMSD for the residue range the superpose calculation based upon. What I wanted is to superpose the two structures with the N-terminal 100 residues, but calculate the RMSD for the entire structure (400 residues). Can LSQKAB do that? Best regards, Huiying Huiying Li, Ph. D Department of Molecular Biology and Biochemistry Natural Sciences I, Rm 2443 University of California at Irvine Irvine, CA 92697, USA Tel: 949-824-4322(or -1953); Fax: 949-824-3280 email: h...@uci.edu On Wed, 24 Nov 2010, Eleanor Dodson wrote: Yes - I think there is a button on the GUI to click? Eleanor On 11/24/2010 01:58 AM, Huiying Li wrote: Another question on RMSD: I have two structures of the same protein superposed with the LSQ Superpose in Coot by matching the first ~100 residues of the N-terminal domain. Now I'd like to calculate the pair-wise RMSD for the entire pre-superposed structures (~400 residues). Can LSQKAB output RMSTAB for the two input PDB files without doing its own FIT/MATCH calculation? Thanks, Huiying ___ Huiying Li, Ph. D Department of Molecular Biology and Biochemistry Natural Sciences I, Rm 2443 University of California at Irvine Irvine, CA 92697, USA Tel: 949-824-4322(or -1953); Fax: 949-824-3280 email: h...@uci.edu
[ccp4bb] protein interaction
Dear All, Could you please provide me some suggestions on following. 1. Could there be difference in interaction between two proteins when produced a) both in bacteria, b) both IVTT (in vitro transcription and translation), and c) one in bacteria and other by IVTT. Could you please direct me to some references. 2. I am looking for references where two proteins not interacting by pulldown or Native gel analysis (or other assays using purified protein) but protein complex crystal structure was solved. Thanks Happy Thanksgiving Yogi
Re: [ccp4bb] protein interaction
It is somehow related to your second question. These folks describe a method for crystallization of low affinity complexes. I hope that helps. Ignatev, A. et al. (2007) A size filtration approach to purify low affinity complexes for crystallization J. Str. Biol. 159(1), 154-157 Cheers, Mario Sanches On Wed, Nov 24, 2010 at 3:11 PM, Sollepura Yogesha yoge...@scripps.eduwrote: Dear All, Could you please provide me some suggestions on following. 1. Could there be difference in interaction between two proteins when produced a) both in bacteria, b) both IVTT (in vitro transcription and translation), and c) one in bacteria and other by IVTT. Could you please direct me to some references. 2. I am looking for references where two proteins not interacting by pulldown or Native gel analysis (or other assays using purified protein) but protein complex crystal structure was solved. Thanks Happy Thanksgiving Yogi -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] protein interaction
Producing the proteins in cell free system or bacterial expression can affect the removal of the start methionine. Although the same rules of a small amino acids next to the start methionine apply, different methionine aminopeptidases tolerate certain small ones better. Depending on the which cell-free kit you use, you may have not removed the methionine, which may be important in protein binding. Protein N-Terminal Processing: Substrate Specificity of Escherichia coli and Human Methionine Aminopeptidases Biochemistry 2010, 49, 5588–5599 A second important consideration is that in cell-free systems other post-translational modifications can be a hit or miss depending on the cell-free kit and/or type of modification. For example acetylation occurs in transdirect insect line N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry PROTEOMICS Volume 6, Issue 16, No. 16 August 2006, Pages: 4486–4495 but not wheat-embryo Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system Protein Expression and Purification 52 (2007) 59–65 though it can be achieved by the addition of Acetyl-CoA.
Re: [ccp4bb] Can LSQKAB calculate RMSTAB for two pre-superposed structures?
On Wed, 2010-11-24 at 09:54 -0800, Huiying Li wrote:
I have two structures of the same protein superposed with the LSQ
Superpose in Coot by matching the first ~100 residues of the
N-terminal
domain. Now I'd like to calculate the pair-wise RMSD for the entire
pre-superposed structures (~400 residues). Can LSQKAB output RMSTAB
for
the two input PDB files without doing its own FIT/MATCH
calculation?
If you save your superposed model in a separate pdb file you can use
this
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Print_the_shifts_in_individual_atom_positions
to get the list of individual atomic shifts. You probably don't care
about all the atoms, so you can just add | grep CA | sort -k3 -n to
the end of the command to get the list of shifts for CA's. So the full
command will look like this
grep 'ATOM\|HETATM' file1.pdb file2.pdb | \
grep -v REMARK | \
cut -d: -f 2 | \
cut -c 13-54 | \
sort | \
awk 'BEGIN {FIELDWIDTHS = 14 28; pt=} {if(pt==$1) print pr,$2; pt=
$1; pr=$0;}' | \
awk 'BEGIN {FIELDWIDTHS = 14 4 8 8 8 5 8 8 8} {printf %s %8.4f\n,
$1,sqrt(($3-$7)^2+($4-$8)^2+($5-$9)^2);}' | \
awk 'BEGIN {FIELDWIDTHS = 4 1 3 1 1 5 9} {printf %s %s %s %s %s\n,
$3,$5,$6,$1,$7;}' | \
grep CA | sort -k3 -n
Cheers,
Ed.
--
I'd jump in myself, if I weren't so good at whistling.
Julian, King of Lemurs
Re: [ccp4bb] unusual diffraction spots
To further clarify things, the data was collected at a synchrotron beamline with collimator size ~130*40(um*square), beam divergence ~0.3*0.1mRad. The detector type was MarCCD. The crystal was multiple-faced trigonal (space group P3121) the size was about 0.1*0.1*0.15mm. The exposure time was 2s for each image. I am currently refining the structure, however the Rfree stays above 30%. A close inspection shows at high resolution shell the spots become rod shaped. As I said we are preparing new constructs with N-terminal his-tag cleaved. But any other good suggestions out there might be helpful to avoid future frustration. Thanks, Hubing On Wed, Nov 24, 2010 at 10:26 PM, elizabeth.d...@diamond.ac.uk wrote: I think there may be two effects going on here: I think the “ears” on the round spots which also feature on the more rod shaped spots if you look closely could be related to a misalignment of the beamline optics. I think the change in spot shape from round to rod shaped is due to the crystal quality. Do the “ears” only feature on this image of this crystal or do they appear on other images? If the ear effect is a one off then that would tend to suggest it isn’t a beamline optic effect. Liz Dr. Liz Duke Principal Beamline Scientist Diamond Light Source Harwell Science and Innovation Campus Chilton OX11 0DE UK Tel. 01235 778057 Mob. 07920 138148 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Hubing Lou *Sent:* 24 November 2010 14:09 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] unusual diffraction spots Dear CCP4BBer, I recently collected a dataset at synchrotron. The diffraction was quite anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What unusual is in the diffraction image (see the attached file), clearly at low resolution there were some spots with tails (two ears) and at the high resolution shell the spots turned to be rod-shaped. Please, can anyone explain how this could be? Is this related to the anisotropy? The protein was N-terminal his6-tagged, we are currently preparing new samples with the His-tag removed. But any other suggestions are also very welcomed. Regards, Hubing -- 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
