Re: [ccp4bb] refining structures with engineered disulfide bonds

[Bart Hazes]

SSBOND actually predicts pairs of residues that, if mutated to cysteine, can form a stable disulfide but does so on first principles, not with a database of preferred conformations. (The server is at http://129.128.24.248/forms/ssbond.html) In this case the 2.7 Angstrom S-S distance is clearly too long for a disulfide (~2.05 A) and too close for a VdW interaction between thiols. There are papers on radiation damage that discuss different configurations that have been observed including their likely chemical nature. In similar cases we have used SHELX in the past to refine occupancy for the different conformations, ensuring the bonded and non-bonded sulfurs and Cbetas in the two cysteines have the same occupancy and the sum for both conformations adds up to 1. There have been several CCP4bb discussion on how to define disulfides in REFMAC but this remains somewhat tricky (at least the last time we needed it) and it does not refine occupancy. Bart On 10-10-20 03:34 PM, Frederic VELLIEUX wrote: Bart Hazes wrote a program (and published as well, Hazes & Dijkstra perhaps) called SSBOND I think. I cannot remember exactly what the computer program does, but it certainly has a data base of "possible" disulphide bond conformers. Hence I would myself certainly check your second didulphide to see if one of these conformers would explain the density better. As long as the model is not into its proper conformation (or conformations if there are several) then the density will not be optimal. There may be SS bond conformer libraries in graphics programs, I do not know (never needed that myself). Fred. > Message du 20/10/10 21:41 > De : "Seema Mittal" > A : CCP4BB@JISCMAIL.AC.UK > Copie à : > Objet : [ccp4bb] refining structures with engineered disulfide bonds > > Hi All, > I have engineered intra-molecular disulfide bond in my protein monomer. The protein functions as a homodimer.  >  In crystal structure, there is clean electron density for the S-S  bond in one monomer (bond length 2.2A), but there seems to be slightly messy density for the same in the other monomer with (S-S bond length of 2.7A). An alternate conformation of one of the cys seems plausible on the messy side. There is considerable negative density associated with this region in both monomers, more so on the messy side.    > My question is :  do i need to select additional parameters or define any sort of constraints during refinement, in order to refine this introduced covalent bond? > Thanks much for your help. > > Best Seema Mittal Graduate Research Assistant Department of Biochemistry & Molecular Pharmacology, > 970L Lazare Research Building, > University of Massachusetts Medical School, > 364 Plantation Street, > Worcester, MA 01605 -- ============================================================================ Bart Hazes (Associate Professor) Dept. of Medical Microbiology & Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax: 1-780-492-7521 ============================================================================