Re: [ccp4bb] Does the substrate has access to the active site?
Hi, I am assuming the enzyme is not active, or by substrate you do not mean the actual substrate, may be an analogue. The substrate might be converted into product and the leave through the channel and you will not find anything bound to it. But I think you have taken care of that. On Fri, Apr 9, 2010 at 7:00 AM, Ed Pozharski wrote: > It is also possible that mother liquor prevents binding (although often > in such cases you would see some precipitant component in the active > site. > > I would generally bet on need for conformational change. And you expect > to see the product complex, right? > > Ed. > > On Fri, 2010-04-09 at 11:15 +0200, Paul Lindblom wrote: > > Dear Bulletin Board, > > > > I am trying to soak substrate into crystals of an enzyme, but so far I > > can't see the substrate in the structure. Does anyone knows a program > > to ensure that the entrance to the central cavity is accessibly? I > > mean based on the whole crystal. I already checked the crystal packing > > manually and it seems that the way is free more or less, but I find it > > hard to interpret. > > > > Thanks in advance, > > > > P. > > > -- > Edwin Pozharski, PhD, Assistant Professor > University of Maryland, Baltimore > -- > When the Way is forgotten duty and justice appear; > Then knowledge and wisdom are born along with hypocrisy. > When harmonious relationships dissolve then respect and devotion arise; > When a nation falls to chaos then loyalty and patriotism are born. > -- / Lao Tse / >
Re: [ccp4bb] Does the substrate has access to the active site?
It is also possible that mother liquor prevents binding (although often in such cases you would see some precipitant component in the active site. I would generally bet on need for conformational change. And you expect to see the product complex, right? Ed. On Fri, 2010-04-09 at 11:15 +0200, Paul Lindblom wrote: > Dear Bulletin Board, > > I am trying to soak substrate into crystals of an enzyme, but so far I > can't see the substrate in the structure. Does anyone knows a program > to ensure that the entrance to the central cavity is accessibly? I > mean based on the whole crystal. I already checked the crystal packing > manually and it seems that the way is free more or less, but I find it > hard to interpret. > > Thanks in advance, > > P. -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Does the substrate has access to the active site?
Paul, couple things come to mind: -make sure your substrate is actually a binder and not an (unspecific) inhibitor (ITC, Thermofluor, Biacore) -soak longer and at higher concentration. (3mM concentration from 100mM stock in DMSO for a week or more) -Is there evidence that your protein needs to undergo substantial rearrangement prohibited by the crystal packing? Then you may be out of luck with soaking, try co-crystallization. The folks from GSK had a nice paper about some useful techniques for ligand incorporation probably a worthwhile read: Acta Cryst. (2007). D63, 72-79 (doi:10.1107/S0907444906047020) HTH Carsten > -Original Message- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of > Paul Lindblom > Sent: Friday, April 09, 2010 5:15 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Does the substrate has access to the active site? > > Dear Bulletin Board, > > I am trying to soak substrate into crystals of an enzyme, but so far I > can't see the substrate in the structure. Does anyone knows a program > to ensure that the entrance to the central cavity is accessibly? I > mean based on the whole crystal. I already checked the crystal packing > manually and it seems that the way is free more or less, but I find it > hard to interpret. > > Thanks in advance, > > P.
[ccp4bb] Does the substrate has access to the active site?
Dear Bulletin Board, I am trying to soak substrate into crystals of an enzyme, but so far I can't see the substrate in the structure. Does anyone knows a program to ensure that the entrance to the central cavity is accessibly? I mean based on the whole crystal. I already checked the crystal packing manually and it seems that the way is free more or less, but I find it hard to interpret. Thanks in advance, P.
