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Definitely Ni, and maybe add another two waters to fill in the density. Regarding B-factors, it depends on those of the surrounding side chains, and should be a bit higher than theirs. Also, since it’s probably not a biologically-relevant Ni site, it would have low affinity and therefore you could plausibly lower the occupancy to make it work (b-factor would decrease.)

 

JPK

 

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of ansuman biswas
Sent: Monday, June 22, 2015 7:04 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement

 

I tried refining with a phosphorylated His (NEP, attached figures 1 and 2 ). After refinement the geometry looks fine, there are no short contacts and the B-factors on the attached phosphate are ~30A2, except for one O-atom which is at 50 A2. However, some positive density is showing up in the Fo-Fc map.

I also tried refining with unmodified His, but with a Ni-ion and 2 water molecules. Ni was present in the crystallization condition. After refinement (3rd Fig attached), there is hardly much positive density in Fo- Fc map. However, the B-factors of the added Ni and water molecules are ~50A2. The Ni coordination site can have both His (predominant) and Lys.

The data was collected at wavelength 0.9A and has resolution 2.3A

 

Please suggest.

Regards,

Ansuman

 

 

On Tuesday, 23 June 2015 1:13 AM, Shane Caldwell <[log in to unmask]> wrote:

 

It's probably much less likely than metal coordination and it's hard to judge from only one angle, but phospho-histidine might be something else to consider.

http://www.jbc.org/content/276/5/3247.full

Shane

 

On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett <[log in to unmask]> wrote:

I agree...one possibility is a M-His(2)-Lys-(OH2) site. Possible metal ions would include Zn(II), although Lys is a relatively rare ligand in zinc-metalloenzyme sites.

Cheers,

_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [log in to unmask]

On 6/22/2015 11:20 AM, Keller, Jacob wrote:

Looks to me like a metal binding site with those histidines, perhaps--any chance of that? That might also explain the weird geometry issues.

JPK

-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Dale Tronrud
Sent: Monday, June 22, 2015 11:17 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] distorted phosphate molecule geometry after refinement

    It is possible that your PO4 has its atoms labeled with the wrong chirality.  Yes, I know that PO4 is not chiral when you ignore hydrogen atoms and single/double bonds but adding labels creates an unnatural chriality.  Try your refinement again after switching the labels on two oxygen atoms.

Dale Tronrud

On 6/22/2015 7:48 AM, ansuman biswas wrote:

Dear CCP4 users,

I am working on a protein from a hyperthermophilic archaeon.

I have collected mutliple X-Ray datasets, both from home source and
synchrotron and always found a clear density for tetrahedral geometry,
co-ordinated by two histidines and one lysine.

I tried fitting phosphate there, but its geometry always gets
distorted after each refinement cycle (Refmac 5.8.0073). Also I found
some short contacts between the coordinated residues and phosphate
which were very difficult to remove.

I am attaching a figure with the density and phosphate.

Kindly suggest -
1. if this may be a possible modification of any of the associated
residues, and the code of the modified residue to be used.

2. If the ligand requires separate restraints during refinement, I am
using the "restrained refinement" option available at the top of the
GUI for refmac.

Thanking you,
yours sincerely,
Ansuman Biswas,
PhD student,
Dept. of Physics,
IISc