Dear Remie, The sugar restraints that come with Refmac should be ok. Even when the sugar is not well defined (weak electron density), the gradients in the electron density maps are usually weak as well and should not distort your bonds. If you have to fix your ligand, or introduce artificially strong restraints, usually the fit is incorrect. What I would do in this case is:
-critically examine the electron density. Would a different orientation (e.g. 180° rotated) or pucker fit better? -fit the sugar manually (rotate/translate zone). To change the pucker, you might have to grab single atoms (cntrl-left mouse button) and move them to another position since the energy barrier between different puckers is often too high for real-space refinement. -do a real space refinement of the sugar alone. Ignore the bond to the Asn/Ser to see if the electron density alone would put the sugar in approximately the correct position. -If your problems are between sugars, you may want to fit each sugar individually and you may need several tries before you find the right orientations. -Of course, if you have clear evidence for multiple conformations, you might have to fit them, but I would only do as a very last resort this if you cannot find a satisfactory major conformation. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Remie Fawaz-Touma Gesendet: Freitag, 15. August 2014 23:28 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] desiring untouched ligand Hi all and thanks for your input. I am trying to fix a sugar chain that has consecutive glucoses and I need the angle C-O-C to be as close to 104 degrees as possible keeping alpha bonds between the sugars, so when I do a Real Space Refinement in Coot, the structure gives angles close enough to 104, and the C-O bonds are at acceptable lengths. Running refmac 5 afterwards always deviates the angles by a great amount to 145 degrees or more for most of the angles. So when I run a Real Space Refinement in Coot for the Refmac5 product, the angles readjust to what is "normal" close to 104. I have tried restrained and rigid body refinement. And I did external harmonic restraints according to suggestions below by creating a pdb file having only the sentence: "external harmonic residues from 843 E to 850 E sigma 0.02", but I still get very wide angles, any suggestion? Thanks for your help, Remie On Aug 14, 2014, at 11:53 AM, Robert Nicholls <nicho...@mrc-lmb.cam.ac.uk<mailto:nicho...@mrc-lmb.cam.ac.uk>> wrote: Hi Remie, You could always generate self-restraints using one chain or a specific part of a chain using prosmart. These will ensure that the model does not move too far from its current conformation. Let me know if you want help to do this. Regards, Rob On 14 Aug 2014, at 16:11, Remie Fawaz-Touma wrote: Thank you Dr. Murshudov for the information. But please I still need help. In restrained refinement, I could not find where to enter the residues I want to restrict from moving much. I was only able to find that in rigid body refinement, so I tried it and got a higher R factor than the initial file (before refinement). Is it normal to get a higher R factor when doing rigid body refinement? if not, any suggestion regarding what could be wrong? Is there a way to restrict one chain or part of a chain from moving much while doing restrained refinement? Thank you for any input, Remie On Aug 13, 2014, at 10:48 AM, Garib Murshudov <ga...@mrc-lmb.cam.ac.uk<mailto:ga...@mrc-lmb.cam.ac.uk>> wrote: Hi Remie, You can add harmonic restraints for parts you do not want to move too much. Instructions could be found here: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html#Harmonic Regards Garib On 13 Aug 2014, at 15:44, Remie Fawaz-Touma <remiefa...@gmail.com<mailto:remiefa...@gmail.com>> wrote: Hi everyone, Does anyone know of a way to refine with CCP4 - Refmac5 (restrained refinement is what I do) fixing a part of the the ligand? Thank you very much for your input, Remie Dr Garib N Murshudov MRC-LMB Francis Crick Avenue Cambridge CB2 0QH UK Web http://www.mrc-lmb.cam.ac.uk<http://www.mrc-lmb.cam.ac.uk/>, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
