On 06/27/2014 06:33 AM, Bernhard Rupp wrote: > For small ion soaking for phasing purposes, partial occupancy is not a > problem. For example, a few 1/2 occupied Iodines still can phase quite well. > 1/2 a C is only 3 electrons, not that great. Add in higher displacement, and > odds are that the ligand interpretation will become difficult. Particularly > when the binding constants are poor, one will out of principle never reach > full occupancy, which further exacerbates the weak density problem. > Patience is definitely a virtue here. > > BR
Here you are starting to mix equilibrium arguments with the previous kinetic arguments. If you have a weak binder you can always get full occupancy by adding enough of the compound - to determine how much, you must consider not only the binding constant but the number of binding sites in the crystal and the total volume of the drop containing your crystal. Time is not a factor. Halide ions and cryoprotectants are known to pervade crystals very rapidly, but they are usually added with "overwhelming force". Much more is added than is required to bind to every specific binding site in the crystal. The rate of diffusion, as mass flow, depends not only on viscosity but on the concentration of unbound molecules inside the crystal. When I was soaking an inhibitor into a crystal of Thermolysin I was having problems with the crystals falling apart. My belief was that the inhibitor caused a small change in cell constants and since the inhibitor first bound in a shell around the surface of the crystal strain was created and the crystal cracked. My solution was to add small aliquots of inhibitor with a long enough wait between to allow each batch to diffuse throughout the crystal. Despite waiting up to 6 hours between additions the crystals still cracked. This is when I realized that after the inhibitor bound in the outer shell of the crystal the remaining concentration of free inhibitor was one billionth (since the binding constant was nanomolar) that of the concentration of active sites and the remaining mass flow within the crystal was insignificant. Of course the next aliquot would rapidly diffuse through the occupied region of the crystal and be bound in the shell just below it, becoming trapped itself and increasing the strain. 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 lack of information makes it very difficult to draw any conclusions from the experiment, but I believe the experience from many other molecules is that small molecules do move very rapidly through protein crystals, until they are caught by a binding site. I don't believe your movie represents typical diffusion of small molecules in a protein crystal. My interpretation of your movie is: 1) The dye rapidly diffuses into the crystal reaching a simple equilibrium where the concentration in the bulk solvent matches that of the outside solution. Since the protein excludes about half of the volume of the crystal the overall concentration is half that of the mother liquor and the color of the crystal is 1/2 as dark as the surrounding solution. 2) With a slow kon, the dye molecules within the crystal start binding specifically to the protein. Since the dye is aromatic it probably has to dig deep into the protein to find a binding site and this takes time. As dye is removed from the bulk solvent it is rapidly replaced by diffusion from outside the crystal, and the crystal begins to darken, eventually becoming darker than the surrounding liquid. The speed of binding is controlling the kinetics not diffusion. Dale Tronrud > > -----Original Message----- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Keller, Jacob > Sent: Friday, June 27, 2014 3:07 PM > To: CCP4BB@JISCMAIL.AC.UK > 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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Bernhard Rupp > Sent: Friday, June 27, 2014 9:00 AM > To: CCP4BB@JISCMAIL.AC.UK > 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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Boaz > Shaanan > Sent: Friday, June 27, 2014 2:26 PM > To: CCP4BB@JISCMAIL.AC.UK > 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: bshaa...@bgu.ac.il > Phone: 972-8-647-2220 Skype: boaz.shaanan > Fax: 972-8-647-2992 or 972-8-646-1710 > > > > > > ________________________________________ > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat > [rkha...@ccny.cuny.edu] > Sent: Friday, June 27, 2014 2:00 PM > To: CCP4BB@JISCMAIL.AC.UK > 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 > = >
