[ccp4bb] few questions about resolving new structure through MR
Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] few questions about resolving new structure through MR
Count yourself lucky that you may have a partial solution for your structure with only 30% identity. The question now is: can you see any reasonable, traceable electron density for domain A? Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 11/7/2013 11:36 AM, Zhihong Yu wrote: Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] few questions about resolving new structure through MR
The fact that you have a 10% split between R/Rfree means your solution is heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55 would imply randomness. So unfortunately in this case, I dont think that you have an actual solution. You could try MR with a poly-A form of the homology model to see if you get a better phaser solution. Then proceed with the refinement while being careful to keep the R/Rfree within 5% and slowly build in the residues of the rest of your protein based on adequate electron density. Hope this helps. - Greg --- Greg Costakes, Ph.D. Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 - Original Message - From: Zhihong Yu nkyuz...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, November 7, 2013 11:36:51 AM Subject: [ccp4bb] few questions about resolving new structure through MR Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] few questions about resolving new structure through MR
First of all, thanks so much for your reply. To Roger: NO, unfortunately I cannot see too much traceable electron density outside the placed atoms, so I think just as Greg said, it's only a model-biased solution. To Greg: YES, I also realized that the input model should be very important, so I'm going to try only backbone of structureX, or build a homology model of domainB first and then put it as a search model. Actually, I asked those questions because I had no idea that even I can correctly place domainB using structureX as a search model, can I really resolve the full length structure? after all, the resolution is only 3.5, and the domainB is only contain 40% residues of the full length. I really want to get some opinions from you expert whether it's worth to spend much time on trying to resolve the strucutre through MR based on current dataset. Or I have to prepare SeMet protein to get experimental phasing information? Thank you all again and look forward to hearing from more expert! Zhihong On Thu, Nov 7, 2013 at 1:25 PM, Greg Costakes gcost...@purdue.edu wrote: The fact that you have a 10% split between R/Rfree means your solution is heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55 would imply randomness. So unfortunately in this case, I dont think that you have an actual solution. You could try MR with a poly-A form of the homology model to see if you get a better phaser solution. Then proceed with the refinement while being careful to keep the R/Rfree within 5% and slowly build in the residues of the rest of your protein based on adequate electron density. Hope this helps. - Greg --- Greg Costakes, Ph.D. Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 -- *From: *Zhihong Yu nkyuz...@gmail.com *To: *CCP4BB@JISCMAIL.AC.UK *Sent: *Thursday, November 7, 2013 11:36:51 AM *Subject: *[ccp4bb] few questions about resolving new structure through MR Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong
Re: [ccp4bb] few questions about resolving new structure through MR
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Zhihong, in many labs a SeMet prep is not much effort and can be done within a week or two, if you express in E.coli. Unless the costs are a limiting factor I would certainly go this way. However, with native data to 3.5A I suggest you contact somebody knowledgeable to help you collect a MAD data set - this will be difficult enough. While the protein is being expressed and purified you can start a couple of MR jobs with different search models - I would use sculptor or mrtailor, though, instead of a plain poly-ALA model: don't make life more complicated than necessary. Regards, Tim On 11/07/2013 08:31 PM, Zhihong Yu wrote: First of all, thanks so much for your reply. To Roger: NO, unfortunately I cannot see too much traceable electron density outside the placed atoms, so I think just as Greg said, it's only a model-biased solution. To Greg: YES, I also realized that the input model should be very important, so I'm going to try only backbone of structureX, or build a homology model of domainB first and then put it as a search model. Actually, I asked those questions because I had no idea that even I can correctly place domainB using structureX as a search model, can I really resolve the full length structure? after all, the resolution is only 3.5, and the domainB is only contain 40% residues of the full length. I really want to get some opinions from you expert whether it's worth to spend much time on trying to resolve the strucutre through MR based on current dataset. Or I have to prepare SeMet protein to get experimental phasing information? Thank you all again and look forward to hearing from more expert! Zhihong On Thu, Nov 7, 2013 at 1:25 PM, Greg Costakes gcost...@purdue.edu wrote: The fact that you have a 10% split between R/Rfree means your solution is heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55 would imply randomness. So unfortunately in this case, I dont think that you have an actual solution. You could try MR with a poly-A form of the homology model to see if you get a better phaser solution. Then proceed with the refinement while being careful to keep the R/Rfree within 5% and slowly build in the residues of the rest of your protein based on adequate electron density. Hope this helps. - Greg --- Greg Costakes, Ph.D. Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 - -- *From: *Zhihong Yu nkyuz...@gmail.com *To: *CCP4BB@JISCMAIL.AC.UK *Sent: *Thursday, November 7, 2013 11:36:51 AM *Subject: *[ccp4bb] few questions about resolving new structure through MR Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.15 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFSe/udUxlJ7aRr7hoRAqMvAJ9Pl9KKQL1Ce56HrNe7wKo+EO2U1gCg4rLF /FqvLWRYfxM3/3MJjX5HyPQ= =RNgU -END PGP SIGNATURE-
Re: [ccp4bb] few questions about resolving new structure through MR
Do you expect more than one molecule in the asymmetric unit? Determined from the Matthews Coefficient (poor), size exclusion column (better), or self RF (best) ? On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.com wrote: Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] few questions about resolving new structure through MR
Thanks Francis, No, only one molecule in the asu. The Matthews Coefficient is 3.3, corresponding solvent content is 62.6%, maybe that's why this crystal show such weak diffraction? Zhihong On Thu, Nov 7, 2013 at 5:37 PM, Francis Reyes francis.re...@colorado.eduwrote: Do you expect more than one molecule in the asymmetric unit? Determined from the Matthews Coefficient (poor), size exclusion column (better), or self RF (best) ? On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.com wrote: Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] few questions about resolving new structure through MR
Dear Debanu, Thanks for your detailed reply. The Z-Score in my current MR trial is only 4.2, which means that domainB was not correctly placed at all, the observed density is indeed model biased density. Since it's my first experience of resolving a new structure, I'm really not sure whether it's worth to put too much efforts on MR based on current 3.5A dataset and only a structure with low homology with one domain. From your reply, I think it's still worth to try a little bit and got information as much as I can. I'm going to try MR Rosetta first. Best Regards! Zhihong On Thu, Nov 7, 2013 at 6:36 PM, Das, Debanu deb...@slac.stanford.eduwrote: Hi Zhihong, The 3.5A diffraction could be due to many reasons: N- and C-term regions, interdomain linker possibly giving rise to molecular flexibility, quality of the particular crystals, cryo, purification, tags, etc. One thing to try is to run secondary structure predictions (or BLAST against PDB, FFAS) on the N- and C-term regions and optimize your construct to exclude some or all of them, especially if you have evidence that they might not be functionally important. 1) Observing density corresponding to your protein sounds promising. What is your PHASER Z-score? Usually Z-scores 8 are indicative of correct solutions so if you are confident that you have the correct placement/solution for domain B, you can try to optimize refinement/model using DEN or MR Rosetta or morph_model. 2) Try the above and see if you can improve your model/maps/R-values. Try optimizing your model (changing residues, removing loops, etc.) by homology modeling (you can try using the PSI Modeling Portal http://www.proteinmodelportal.org/) or other similar services or try different programs individually. In addition, try to obtain a homology model of domainA (including model building with Rosetta/Robetta). Additional phasing information by experimental phasing using SeMet or heavy atoms will be best, but is often easier said than done. Since you are at the MR stage, it will be useful if you can squeeze as much information as you can from MR efforts. If you are sure you have domainB placed correctly (and can also obtain a reliable solution for domainA), your MR phases can be used later on to locate heavy atom sites by difference Fourier methods and you can also combine with experimental phases in non-optimal cases Best, Debanu. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zhihong Yu [nkyuz...@gmail.com] Sent: Thursday, November 07, 2013 2:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] few questions about resolving new structure through MR Thanks Francis, No, only one molecule in the asu. The Matthews Coefficient is 3.3, corresponding solvent content is 62.6%, maybe that's why this crystal show such weak diffraction? Zhihong On Thu, Nov 7, 2013 at 5:37 PM, Francis Reyes francis.re...@colorado.edu mailto:francis.re...@colorado.edu wrote: Do you expect more than one molecule in the asymmetric unit? Determined from the Matthews Coefficient (poor), size exclusion column (better), or self RF (best) ? On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.commailto: nkyuz...@gmail.com wrote: Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] If it is a new structure?
Dear CCP4 users, I would like to draw your attention to the topic If it is a new structure?, which is one more case, when new community members were welcomed, and were gently explained about the rules of files sharing, as it is an evidence, that the rules about files sharing are not well defined and highlighted as a crucial point (as it appears to be) during the subscription. I think, that proper introduction of these rules on the page of subscription is much more easier than to explain them to every new member who posts files not properly. Best, Sergii On 12/21/2010 01:02 AM, CCP4BB automatic digest system wrote: You're welcome! Next time, irrespective of whether your structure is a new one or not, please upload your images to picasa, flickr, your university's file sharing server or some such thing and include the link in your email. Don't flood several thousand inboxes with megabytes of pixels. And compress bitmaps, for crying out loud. How hard can it be? Thanks. Andreas On 20/12/2010 10:49,Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help. -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- Sergii Buth PhD student of Prof. Petr Leiman Laboratory of Structural Biology and Biophysics Institut de physique des systèmes biologiques École Polytechnique Fédérale de Lausanne (EPFL) Cubotron/BSP-416 CH-1015 Lausanne Switzerland Phone: +41 21 693 04 40
Re: [ccp4bb] If it is a new structure?
Hi, Last couple of times I asked myself the same question (what does it look like?) I used ssm (or PDBeFold as seems to be called now). http://www.ebi.ac.uk/msd-srv/ssm/ HTH, Fred. Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help.
Re: [ccp4bb] If it is a new structure?
You're welcome! Next time, irrespective of whether your structure is a new one or not, please upload your images to picasa, flickr, your university's file sharing server or some such thing and include the link in your email. Don't flood several thousand inboxes with megabytes of pixels. And compress bitmaps, for crying out loud. How hard can it be? Thanks. Andreas On 20/12/2010 10:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help. -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] If it is a new structure?
Hi Liu, If I understand your question correctly, youre asking how different do two structures need to be for one to be new'. If by new you mean a new fold, then the answer is NO. Your structure and the homolog have the same fold. However, if your structure is the first structure of a protein in a new class, then your structure is a new insight for that reason (e.g. it is the first structure of a Unobtainium-metalloprotease). If it is not the first structure of a protein from a new class, lets say a previous structure of Unobtainium-metalloprotease has been solved using H. sapiens' sequence, but your protein is the first D. melanogaster ortholog solved, then your structure is a new insight for that reason. So, in a nut-shell, I guess what I am saying is that your protein is not a new fold, but is almost certainly new by some qualification, and you will know best what that qualification is. I hope that helps, cheers and happy holidays~ ~Justin On 20/12/2010 10:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help.
Re: [ccp4bb] If it is a new structure?
On 12/20/10 05:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help. The structure appears to consist of 3 distinct domains, a central beta barrel and two mostly alpha helical domains. RE the failure of molecular replacement - you don't say how hard you tried. Given a starting structure like that, I would have tried searching separately with models for the various domains. I also cannot tell from two images how similar the helical domains are; is it just a simple rotation, or is the arrangement of helices substantially different? There are algorithms for comparing protein folds. This article addresses your need, but is a bit old: http://www.ncbi.nlm.nih.gov/pubmed/14696188 Evaluation of protein fold comparison servers. Novotny M http://www.ncbi.nlm.nih.gov/pubmed?term=%22Novotny%20M%22%5BAuthor%5D, Madsen D http://www.ncbi.nlm.nih.gov/pubmed?term=%22Madsen%20D%22%5BAuthor%5D, Kleywegt GJ http://www.ncbi.nlm.nih.gov/pubmed?term=%22Kleywegt%20GJ%22%5BAuthor%5D. Proteins. 2004 Feb 1;54(2):260-70. PMID: 14696188 [PubMed - indexed for MEDLINE] -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] If it is a new structure?
Hi Liu Looks like (on the images you show) that you have a nice conformational difference for some of the helices between the 2 structures... Happy Hols Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Justin Hall Sent: Monday, December 20, 2010 8:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] If it is a new structure? Hi Liu, If I understand your question correctly, youre asking how different do two structures need to be for one to be new'. If by new you mean a new fold, then the answer is NO. Your structure and the homolog have the same fold. However, if your structure is the first structure of a protein in a new class, then your structure is a new insight for that reason (e.g. it is the first structure of a Unobtainium-metalloprotease). If it is not the first structure of a protein from a new class, lets say a previous structure of Unobtainium-metalloprotease has been solved using H. sapiens' sequence, but your protein is the first D. melanogaster ortholog solved, then your structure is a new insight for that reason. So, in a nut-shell, I guess what I am saying is that your protein is not a new fold, but is almost certainly new by some qualification, and you will know best what that qualification is. I hope that helps, cheers and happy holidays~ ~Justin On 20/12/2010 10:49, Liu Zhao wrote: The structure of my protein is as shown as the purple one. Another one ,as shown as green,is homologous .But the structure of my protein can't be obtained by using molecular replacement. And both structures have much different, especially in B chain. If my structure is a new one? thank you for help. Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
