Hi Again,

Thanks for the suggestions. I just tried it at 2 ligand concentrations and the crystals seem not to mind. The drop does go a little cloudy but I can't say if this is protein precipitation, or the ligand coming out of solution. My feeling its a bit of protein because of the high DMSO conc. being added. I'll co-crystallize just to have a backup!

Cheers

C 

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Christopher Browning, Ph. D

Vertex Pharmaceuticals (Europe) Ltd

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From: <Noinaj>, "Nicholas [E] (NIH/NIDDK)" <[log in to unmask]>
Date: Thursday, 12 September 2013 10:21
To: Christopher Browning <[log in to unmask]>, "[log in to unmask]" <[log in to unmask]>
Subject: RE: Adding ligand to crystallization drop

Chris,

 

Bottom line is that it is all dependent on how your ligand interacts with your protein, if there are any conformational changes, and how your crystals behave in such a dilution and high concentration of ligand.  You just have to test it and see, no way to know for sure until you do the experiments and see.  However, there are some things to look for along the way.  For example, do your crystals crack after introducing the ligand or do they start to dissolve and lose their nice edges, or turn opaque?  you will probably also want to do a buffer only control without ligand to ensure that the crystals are ok with the dilution and change of environment. 

 

I assume the native crystals diffract well enough to solve the structure.  You would want to test crystals before any soaking and then with buffer only and then with ligand, to ensure diffraction is retained to a level useful enough for structure determination.  I did quite a bit of crystal soaking in grad school and it is something you just have to try since every crystal/protein is different. my experience is that soaking can often lead to a slight loss of resolution but typically you still get the information you are seeking if the crystals survive.

 

with that being said, soaking can sometimes lead to partial occupancy of your ligand.  So if you are able to do co-xtallization, i think that would be preferred and doesn't seem like too much additional effort assuming you are getting crystals in the same conditions as with native protein only.  and with co-xtallization, I often see slightly better resolution that with native protein only (not always the same condition though unfortunately).  But again, no way to know how your particular protein/crystal will behave without doing some pilot experiments first. 

 

Good luck!

 

 

 

 

 

Cheers,

Nick

 

 

 

--------------------------------

 

[ Nicholas Noinaj ]

the Buchanan Lab

Laboratory of Molecular Biology

LMB-NIDDK, NIH

50 South Drive, Room 4505

Bethesda, MD  20892-8030

1-301-594-9230 (lab)

1-859-893-4789 (cell)

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[ the Buchanan Lab ]

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From: Christopher Browning [mailto:[log in to unmask]]
Sent: Thursday, September 12, 2013 4:37 AM
To: [log in to unmask]
Subject: [ccp4bb] Adding ligand to crystallization drop

 

Hi,

 

I've just got a quick question about getting ligands bound to proteins in crystals. I've managed to co-crystallize my proteins with the various ligands, and I'm aware that soaking will work as well but I want to speed up the process.

 

Would this work? If I had a bunch of crystals that have grown in their drop, can I pipette the ligand (say 0.6ul to get a desired conc.) onto the drop and just let the crystals soak that way. Any dilution that would have occurred when then readjust as the drop will be sealed again. Perhaps the crystals would get a little stressed when adding the high concentrated ligand and this method would not work…?

 

Thanks,

 

Chris

 

 

 

-- 

Christopher Browning, Ph. D

 

Vertex Pharmaceuticals (Europe) Ltd

86-88 Jubilee Avenue

Milton Park

Abingdon

Oxfordshire

OX14 4RW

United Kingdom

 

Tel +44 (0) 1235 438327

[log in to unmask]

www.vrtx.com

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