Re: [ccp4bb] Cross-validation when test set is miniscule

[Axel Brunger]

Dear Derek,

I suggest you try 10% for the test set.  You should still be able to judge the 
effect of 
various restraints (or constraints) as long as you keep the same test set.  If 
you switch test sets, and re-refine, Rfree
might change as much as 2% for a test set consisting of 200 reflections - see 
Fig. 6 in  ref. (A. T. Brunger, Free  
R value: Cross-validation in crystallography, Methods in Enzym. 277, 366-396, 
1997). However, using the 
same test set may allow you to judge the best restraints protocol or weights. 

Axel

PS: The Methods in Enzym. review also briefly discusses "complete 
cross-validation".

PPS: For refinement at very low resolution, see also:

A.T.Brunger, P.D.Adams, P.Fromme, R.Fromme, M.Levitt, G.F. Schroder. Improving 
the accuracy of 
macromolecular structure refinement at 7 A resolution. Structure 20, 957-966 
(2012).


> On Dec 20, 2014, at 1:05 AM, CCP4BB automatic digest system 
>  wrote:
> 
> Date:Fri, 19 Dec 2014 11:18:37 +
> From:Derek Logan 
> Subject: Cross-validation when test set is miniscule
> 
> Hi everyone,
> 
> Right now we have one of those very difficult Rfree situations where it's 
> impossible to generate a single meaningful Rfree set. Since we're in a bit of 
> a hurry with this structure it would be good if someone could point me in the 
> right direction. We have crystals with 1542 non-H atoms in the asymmetric 
> unit that diffract to only 3.6 Å in P65, which gives us a whopping 2300 
> reflections in total. 5% of this is only about 100 reflections. Luckily the 
> protein is only a single point mutation of a wild type that has been solved 
> to much better resolution, so we know what it should look like and I simply 
> want to investigate the effect of different levels of conservatism in the 
> refinement, e.g. NCS in xyz and B, group B-factors, reference model, 
> Ramachandran restraints etc. However since the quality criterion for this is 
> Rfree I'm not able to do this.
> 
> I believe the correct approach is k-fold statistical cross-validation, but 
> can someone remind me of the correct way to do this? I've done a bit of 
> Googling without finding anything very helpful.
> 
> Thanks
> Derek
> 
> Derek Logan tel: +46 46 222 1443
> Associate Professor mob: +46 76 8585 707
> Dept. of Biochemistry and Structural Biology  
> www.cmps.lu.se
> Centre for Molecular Protein Sciencewww.maxlab.lu.se/crystal
> Lund University, Box 124, 221 00 Lund, Sweden   www.saromics.com

Axel T. Brunger
Investigator,  Howard Hughes Medical Institute
Professor and Chair, Dept. of Molecular and Cellular Physiology
Stanford University

Web:http://atbweb.stanford.edu
Email:  brun...@stanford.edu  
Phone:  +1 650-736-1031

Re: [ccp4bb] non-specific disulfide bonds in crystal structures

[Reza Khayat]

Hi Todd,

A concern of mine is that you may be looking at an artifact 
of the domain. Whatever assays you decide to do, make sure 
that they are also performed for the full sized protein.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
>Date: Sat, 20 Dec 2014 10:44:25 -0800
>From: CCP4 bulletin board  (on 
behalf of Christine Gee )
>Subject: Re: [ccp4bb] non-specific disulfide bonds in 
crystal structures  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Hi Todd,
>   I used to work on PNMT which also is supposedly
>   monomeric and formed a disulfide between monomers in
>   the crystals.
>See 
http://www.sciencedirect.com/science/article/pii/S157096390
5000968?via=ihub
>   We showed that it was irrelevant to activity.
>   Cheers
>   Christine
>   Sent from my iPad
>   On 20 Dec 2014, at 9:52 am, Todd Jason Green
>wrote:
>
> Hello All-
>
> I have recently determined a domain structure of a
> larger protein. The structure shows a clear
> disulfide bond between two monomers in the
> asymmetric unit. I'm trying to figure out if this
> is an artifact of the crystal packing or has
> biological relevance. The protein has been
> reported to function as a monomer. If I look at
> the pool of protein on a SDS-PAGE gel under
> non-reducing conditions, I see that a smaller
> percentage (~15-20%) of the protein runs as a
> dimer. In the structure, the association has
> 2-fold symmetry with about 29% of the monomeric
> surface area buried between the dimer. Can anyone
> point me in the direction of a paper describing a
> non-specific disulfide in a crystal, or perhaps a
> criteria for assessing specificity? I will do some
> functional studies, but I'm looking for some info
> on a lazy saturday.
>
> Thanks in advance. Best-
> Todd

Re: [ccp4bb] non-specific disulfide bonds in crystal structures

[Christine Gee]

Hi Todd,

I used to work on PNMT which also is supposedly monomeric and formed a 
disulfide between monomers in the crystals.  See 
http://www.sciencedirect.com/science/article/pii/S1570963905000968?via=ihub
We showed that it was irrelevant to activity.

Cheers
Christine

Sent from my iPad

> On 20 Dec 2014, at 9:52 am, Todd Jason Green  wrote:
> 
> Hello All-
> 
> I have recently determined a domain structure of a larger protein. The 
> structure shows a clear disulfide bond between two monomers in the asymmetric 
> unit. I'm trying to figure out if this is an artifact of the crystal packing 
> or has biological relevance. The protein has been reported to function as a 
> monomer. If I look at the pool of protein on a SDS-PAGE gel under 
> non-reducing conditions, I see that a smaller percentage (~15-20%) of the 
> protein runs as a dimer. In the structure, the association has 2-fold 
> symmetry with about 29% of the monomeric surface area buried between the 
> dimer. Can anyone point me in the direction of a paper describing a 
> non-specific disulfide in a crystal, or perhaps a criteria for assessing 
> specificity? I will do some functional studies, but I'm looking for some info 
> on a lazy saturday.
> 
> Thanks in advance. Best-
> Todd

[ccp4bb] non-specific disulfide bonds in crystal structures

[Todd Jason Green]

Hello All-

I have recently determined a domain structure of a larger protein. The 
structure shows a clear disulfide bond between two monomers in the asymmetric 
unit. I'm trying to figure out if this is an artifact of the crystal packing or 
has biological relevance. The protein has been reported to function as a 
monomer. If I look at the pool of protein on a SDS-PAGE gel under non-reducing 
conditions, I see that a smaller percentage (~15-20%) of the protein runs as a 
dimer. In the structure, the association has 2-fold symmetry with about 29% of 
the monomeric surface area buried between the dimer. Can anyone point me in the 
direction of a paper describing a non-specific disulfide in a crystal, or 
perhaps a criteria for assessing specificity? I will do some functional 
studies, but I'm looking for some info on a lazy saturday.

Thanks in advance. Best-
Todd