Dear Bernard,
we once worked with a series of protease inhibitors which turned out to be slow substrates, e.g. an acyl intermediate was formed that was subsequently hydrolyzed. Here we had to reduce the soaking times to below 30 minutes, otherwise we would see nothing, e.g. the large excess of added inhibitor was completely turned over. The precipitant was 25% PEG4000, which I consider a typical PEG condition. The inhibitors were the usual bunch of a few (aromatic) rings linked (unfortunately) by an amide linker.
I agree with the others who reacted to your post that soaking times of 10 hrs are atypical and more likely caused by a slow Kon than by slow diffusion.
Cheers,
Herman
-----Ursprüngliche Nachricht-----
Von: CCP4 bulletin board [mailto:[log in to unmask]] Im Auftrag von Bernhard Rupp
Gesendet: Samstag, 28. Juni 2014 10:46
An: [log in to unmask]
Betreff: Re: [ccp4bb] Solvent channels
> Here you are starting to mix equilibrium arguments with the previous
kinetic arguments.
I don't think I am mixing them; both are relevant. If it cannot diffuse there, forget the kinetics - necessary but not sufficient requirement.
Nonetheless, the fact that in high concentrations you can force even weak non-native binders into binding sites (but I reiterate, never in 100% occupancy, at best asymptotically approaching it) is the reason for the many buffer 'ligands' observed in structures (also basis for fragment screening.)
> Your movie doesn't include any details of concentration of your dye,
> nor
what its binding constant is to any sites in a protein nor any mention of kon or koff.
The movie does not claim to be a study of any specific ligand binding, it simply illustrates soaking. Graphs of concentration vs achievable equilibrium occupancy at different Kds are in separate figures eg 3-40.
Cheers, BR
Dale Tronrud
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Keller, Jacob
> Sent: Friday, June 27, 2014 3:07 PM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Solvent channels
>
> ....And yet halides--even iodide--permeate those same lysozyme
> crystals and others entirely in <30--60 sec.
>
> JPK
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Bernhard Rupp
> Sent: Friday, June 27, 2014 9:00 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Solvent channels
>
> Just a remark: diffusion is a slow and random-walk process.
> Particularly large molecules in viscous media (PEG anybody?) move
> (diffuse) slowly in solution. To simply extrapolate from the fact that
> the ligand is smaller than the solvent channels to the odds of the
> presence of a ligand is a risky proposition. Positive omit difference
> density after 'shoot first' as Boaz indicated is a much better indication.
And shoot you probably will a lot.
>
> The little movie below shows how slowly even a small aromatic dye
> molecule soaks into a crystal. Total time 10 hrs.
>
> http://www.ruppweb.org/cryscam/lysozyme_dye_small.wmv
>
> The literally hundreds of empty ligand structures collected in
> Twilight attest to that fact.
>
> http://journals.iucr.org/d/issues/2013/02/00/issconts.html
>
> Best, BR
>
> Science is a way of trying not to fool yourself: The first principle
> is that you must not fool yourself - and you are the easiest person to
fool.
>
> R. Feynman, 1974
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Boaz Shaanan
> Sent: Friday, June 27, 2014 2:26 PM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Solvent channels
>
> Hi,
>
> I'm not aware of a program with an option to display channels in
> crystals but if you use any of the currently available molecular
> display program and ask to display symmetry-related molecules +
> adjacent unit cells, it should give you a good enough idea of the
> spaces between molecules. Using programs for calculation of
> intermolecular
distances would also be helpful here.
> Independently of the calculation, I would try soaking first and
> consult the calculations later (in the spirit of Rossmann's American
> method: shoot first ask later).
>
> Cheers,
>
> Boaz
>
>
> Boaz Shaanan, Ph.D.
> Dept. of Life Sciences
> Ben-Gurion University of the Negev
> Beer-Sheva 84105
> Israel
>
> E-mail: [log in to unmask]
> Phone: 972-8-647-2220 Skype: boaz.shaanan
> Fax: 972-8-647-2992 or 972-8-646-1710
>
>
>
>
>
> ________________________________________
> From: CCP4 bulletin board [[log in to unmask]] on behalf of Reza
> Khayat [[log in to unmask]]
> Sent: Friday, June 27, 2014 2:00 PM
> To: [log in to unmask]
> Subject: [ccp4bb] Solvent channels
>
> Hi,
>
> I'd like to do some soaking experiments with a relatively large molecule.
> Can someone suggest a program/method to display the solvent channels
> of a crystal? We have the crystal structure. I'd like to see if the
> channels are large enough to allow the molecule to travel to the
> hypothesized binding site.
> Thanks.
>
> 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
> =
>
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