Hi Robbie Thanks for useful info. I perhaps didn't express the physiological pH question that well: what was going through my mind is that I assume that in many cases the crystallisation conditions are chosen to be close to the physiological ones simply because one obviously wants to know the biologically relevant structure. I'm aware that in many cases one does not have the luxury of choosing the pH on this basis, but rather the set of buffer conditions found by screening is the only one that produces crystals!
Cheers -- Ian > -----Original Message----- > From: rjoos...@cmbi.ru.nl [mailto:rjoos...@cmbi.ru.nl] > Sent: 28 July 2009 11:08 > To: Ian Tickle > Cc: ccp4bb@jiscmail.ac.uk > Subject: Re: [ccp4bb] HIS restraint target values & SUs. > > Hi Ian, > > > All, I know Clemens Vonrhein raised a similar question about the HIS > > restraint target values & SUs in the standard library some years ago. > > The values currently in use appear to be the Engh & Huber (1991) ones > > for the doubly-protonated (+1 charge) imidazole side-chain, so I have 4 > > questions: > > 1) Shouldn't we be using the E&H (1999) values by now? > Obviously, we should. The makers of refinement programs should update > their restraint libraries if they haven't done so already. > > > 2) Is the doubly-protonated form the most appropriate, given that the > > pKa is supposed to be ~ 6 (I'm aware that the physiological > > extracellular pH of ~ 7.4 may not be relevant inside a protein)? > Notwithstanding the biological question we want to answer, shouldn't we > base this on the crystallisation conditions? You could of course argue > that pH is a statistical property and does not necessarily apply to a > local position in the protein. In that case the neutral form seems to me > the most appropriate form. > > > 3) Do users have the option to use the parameters for the other forms > > (assuming of course they can work out for each case which form is > > present, and therein of course may lie the rub!)? E&H list values for 3 > > forms of HIS: 'HISE' with ND1 unprotonated, NE2 protonated; 'HISD' with > > ND1 protonated, NE2 unprotonated; and 'HISH' the doubly protonated form > > which appears to be the default. > WHAT_CHECK has had a check to find the proper form of HIS for ages. It > considers both the geometry (which only works when the restraints ar not > too tight) and the hydrogen bond network. > > > 4) Would a suitably weighted average of all 3 forms be more appropriate, > > with suitable inflation of the SUs, given that the form actually present > > is likely to be a mixture of tautomeric and/or > > resonance-charged/uncharged forms anyway? > This can vary locally. On many positions in the protein the protonation > state of HIS won't change all that much because it would be energetically > unfavourable. > > Cheers, > Robbie > Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
