-----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 <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 >> > - -- 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-----
