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.edu>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 <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.com<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 > > > > > > >
