Hi Chris,

I tried to refine with Ca++ but density became highly negative. I could only refine it with Mg++ and Na+ without any green or red density. Na+ is less likely because Na+-water distance normally 2.7 - 2.8 Å. The mg++ does not have any interaction with the amino acid residues directly but waters coordinated with Mg++ have hydrogen bonding interaction with protein residues.

I have to analyze structure more carefully and come up with an explanation.

-Rajesh

   

On Tue, Mar 6, 2018 at 8:37 PM, Christopher Squire <[log in to unmask]> wrote:

At 200 mM CaCl2 (I think you said) I'd be surprised if this was not calcium. The distances compared to a dictionary are not always perfect. Put both a Mg(H2O)6 and a Ca(H2O)6 in place and refine. The temperature factors and/or residual electron density in maps will likely tell you which is the case. We have similar issues in some of our structures where the same site can bind both Ca and Mg but the crystallisation is in high concentrations of Ca so most likely calcium not magnesium. Otherwise, if the site is a very specific and strong magnesium binding site then it is possible that Mg is retained from earlier in your purification or even carried over from the bacterial expression medium.

Chris.

Dr. Christopher Squire

Associate Professor, School of Biological Sciences

University of Auckland

Ph: +64 9 923 3086

Ph: internal ext 83086

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From: CCP4 bulletin board <[log in to unmask]> on behalf of Rajesh Kumar <[log in to unmask]>
Sent: 07 March 2018 1:07 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] Unknown density

 

Dear All,

It is mg++ with 6 waters. As mg-water distances are 2 - 2.1 Å. Now from where I got the mg++ in the structure? We have not used Mg++ in purification and no Mg++ in the crystallization condition. 

But the inhibitor, which I used for the co-crystallization might have mg++. Because during drug synthesis, very often we use MgSO4 in drying steps. It might be coming from there.

Thank you very much all,

Rajesh

On Tue, Mar 6, 2018 at 6:57 PM, Prem Prakash <[log in to unmask]> wrote:

Hi Rajesh, 

It might be water molecules present at the symmetry axis, one more water molecule at the centre (exactly at the axis, with occupancy of 0.5). If you put three water molecule you probably may find the other three symmetry-mates  water molecules in opposite sides. OR One water molecule at the symmetry axis.surrounded by 5 different water molecules. Or You can try As Garib mentioned it may be a six anions (or water) coordinated with some metals possibly in crystallization or protein buffer. Please try refining both cases parallely. I once encountered such case in my structure. Probably it may help.

Good luck

Prem 

On Wed, Mar 7, 2018 at 4:37 AM, Abhik Mukhopadhyay <[log in to unmask]> wrote:

Hi Rajesh,

You may find this database is also useful while refining your model

http://mespeus.bch.ed.ac.uk/MESPEUS_10/

and for calcium

http://mespeus.bch.ed.ac.uk/MESPEUS_10/_5.jsp

Abhik

On Tue, Mar 6, 2018 at 10:59 PM, Patrick Loll <[log in to unmask]> wrote:

Calcium likes to form octahedral complexes with water (or other oxygen-containing) ligands. This looks like a classic example.

After you model and refine this, you’ll want to check water-metal distances, to make sure they are appropriate for calcium. There is a nice literature on such things, which I of course don’t have at my fingertips; but I think Wladek Minor has done some data-mining in metal-containing protein structures, and Amy Katz and Jenny Glusker have a number of papers that are relevant. There are more, of course—a little time in the “library” is warranted.

Cheers,

Pat Loll

> On 6 Mar 2018, at 5:19 PM, Rajesh Kumar <[log in to unmask]> wrote:
>
> Dear All,
>
> Have you had experience with this kind of density? I am wandering what this could be?
>
> Thank you very much for the help.
>
> -Rajesh
>
>
> <Screen Shot 2018-03-06 at 5.15.20 PM.png>
>

---------------------------------------------------------------------------------------
Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[log in to unmask]
[log in to unmask]

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With kind regards,

Prem Prakash

PhD Research Scholar

Protein Crystallography Lab

Biosciences and Bioengineering

IIT Bombay