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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 <[log in to unmask]> 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" <[log in to unmask]> *To:
>> *[log in to unmask] *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
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