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 wrote: > 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 <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.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
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 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 mailto: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 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 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
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 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
-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 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" *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
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 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" > *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
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" 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
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
[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
