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 [[email protected]] On Behalf Of Zhihong Yu [[email protected]] Sent: Thursday, November 07, 2013 2:53 PM To: [email protected] 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 <[email protected]<mailto:[email protected]>> 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 <[email protected]<mailto:[email protected]>> 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
