[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
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
[ccp4bb] The density shown in Pymol and Coot is different.
Hi,all I refined a protein-ligand complex structure using Refmac5 and got the map coefficient A.mtz file. I want to represent the electron density in Pymol, so I transformed A.mtz into A.map ccp4-format map file using FFT in ccp4i (parameters are: generate simple map in CCP4 format to cover asymmetric unit, all others are default). When I load A.map inot Pymol and show eletron density around the ligand, I found the electron density was somewhat different comparing to that shown in coot using A.mtz, density in Pymol (using A.map) was much stronger than that in Coot (using A.mtz) when showing at 1.0 sigma level. Where does the differenc come from? Should I set any other parameters when I transform mtz to map using FFT program? Thanks in advance! Zhihong
Re: [ccp4bb] The density shown in Pymol and Coot is different.
Hi, Nian, Thanks for your reply. I figured out the problem just now. The difference indeed came from my FFT transformation. I just used all default parameters when I transform A.mtz to A.map, so in the Run FFT dialog, F1= F, Sigma=SIGF, PHI=PHIC, Weight=Unaasigned, and I got A.map under these conditions. Even when I load A.map into Coot, the density is different with A.mtz. But when I assigned parameters as following: F1= FWT, Sigma=Unaasigned, PHI=PHWT, Weight=Unaasigned, and ran the job, I got another A1.map, the density is exactly same as A.mtz this time whatever in Pymol or Coot. I'm a rookie in this field, so herein I have another question about this: Which map should I use for showing the final density, A.map or A1.map? What's the difference of these two maps, it looks like A.map is much stronger than A1.map. Really thanks for response! Zhihong 2011/1/7 Nian Huang huangn...@gmail.com It has been discussed before. Nothing to do with FFT. Both pymol and coot will rescale your map but differently. You have to really look into how the programmer wrote their program to know what is going on. For now, I suggest you just use coot map which is more realistic to me. Also new version of pymol seems to work better. On Fri, Jan 7, 2011 at 1:42 PM, Zhihong Yu nkyuz...@gmail.com wrote: Hi,all I refined a protein-ligand complex structure using Refmac5 and got the map coefficient A.mtz file. I want to represent the electron density in Pymol, so I transformed A.mtz into A.map ccp4-format map file using FFT in ccp4i (parameters are: generate simple map in CCP4 format to cover asymmetric unit, all others are default). When I load A.map inot Pymol and show eletron density around the ligand, I found the electron density was somewhat different comparing to that shown in coot using A.mtz, density in Pymol (using A.map) was much stronger than that in Coot (using A.mtz) when showing at 1.0 sigma level. Where does the differenc come from? Should I set any other parameters when I transform mtz to map using FFT program? Thanks in advance! Zhihong
Re: [ccp4bb] The density shown in Pymol and Coot is different.
Matthew Nat, Thanks very much, your answers made me much clear about the map manipulating, as well as usage of Refmac and FFT. Just from my results, I think I agree with Nat that Sigma and Weight is unnecessary when running FFT based on a Refmac-resulted mtz file, since my A1.map is exactly same as FWT of A.mtz in Coot. Again thanks for your help! Zhihong 2011/1/7 Nat Echols nathaniel.ech...@gmail.com On Fri, Jan 7, 2011 at 2:34 PM, Matthew Franklin mfrank...@nysbc.orgwrote: However, I'm not sure if you're doing the right thing by leaving SIGF unassigned. I believe this should be assigned to the experimental error, which is usually called 'SIGF' in your mtz file. I don't know if FFT actually uses this for anything when it's calculating your map file. I'm also not sure what you should use for the weight parameter - perhaps 'FOM'? FWT,PHWT has the weights already applied to the amplitudes*, so no FOM is necessary. According to the FFT documentation, the sigmas can be used to exclude reflections (subject to an additional keyword), but I assume by default it will just be ignored. -Nat (* FWT = 2mFo-DFc, where m is the FOM.)