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 for 3 cycyles, 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
On Thu, Nov 7, 2013 at 8:34 AM, Zhihong Yu
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 for 3 cycyles, 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?
No, that's a classic example of model bias. Positive (and continuous) mFo-DFc density outside of the placed atoms is what you should be looking for, but I suspect there will be very little. It's basically impossible to tell from this result whether the placement of structureX is even correct, although the Phaser statistics may be informative. One unrelated suggestion: given the low sequence identity, I think it will be very important to properly prepare the input model (use Sculptor for this). 2. If it’s possible, what’s the general optimal procedure I should follow?
I confess I haven't had to solve a structure like this myself - just seen a bunch of datasets where similar attempts didn't work - but it's hard to see how it can be solved without experimental phasing information (SeMet, etc.). The good news is that if you can get a correct MR solution for domainB, you can use MR-SAD to find heavy atom sites and solve the structure, which should be easier than starting from no phases at all. -Nat
Dear Nathaniel, Thanks for your reply. So from your answer, I feel that maybe it's not worth to spend much time to resolve the structure through MR based on current dataset? Even I can correctly place the domainB, it's still hard to resolve the intact protein structure without other phasing information, is that right? Thanks again and best regards! Zhihong
I confess I haven't had to solve a structure like this myself - just seen a bunch of datasets where similar attempts didn't work - but it's hard to see how it can be solved without experimental phasing information (SeMet, etc.). The good news is that if you can get a correct MR solution for domainB, you can use MR-SAD to find heavy atom sites and solve the structure, which should be easier than starting from no phases at all.
-Nat
On Thu, Nov 7, 2013 at 10:44 AM, Zhihong Yu
Thanks for your reply. So from your answer, I feel that maybe it's not worth to spend much time to resolve the structure through MR based on current dataset? Even I can correctly place the domainB, it's still hard to resolve the intact protein structure without other phasing information, is that right?
There's no absolute rule, but it's extremely difficult even with higher resolution data. Sometimes density modification would help but I suspect the phases just won't be good enough. With an R-free of 0.55 it is very unlikely that you'll be able to see anything interpretable in the maps, even if the model placement is correct. -Nat
participants (2)
-
Nathaniel Echols -
Zhihong Yu