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 >
