On 04/24/2014 08:33 AM, Phoebe A. Rice wrote: > If you are doing real-space refinement of the entire model against a map in > coot, isn't it also important to make sure that the Rfree reflections were > NOT used in calculating that map? Depending on how the map was made, that > info can be hidden in the fine print. > > ++++++++++++++++++++++++++++++++++++++++++ Not sure. Although this would seem to be the real-space analog of omitting the free reflections during reciprocal space refinement, there is actually a big difference. In reciprocal space, the free reflections can be simply ignored. In real space refinement against a map from which the Free reflections were omitted, you are still using the free reflections but with an incorrect value: zero.
Then, if real space refinement really can bias a model to best-fit the reflections, all the free reflections will tend toward zero and your R-free for the model will go up. Then when you compare R-free after RS refinement with or without free reflections, you might incorrectly attribute the difference to bias when using them! eab > > Phoebe A. Rice > Dept. of Biochemistry & Molecular Biology > The University of Chicago > > 773 834 1723; pr...@uchicago.edu > http://bmb.bsd.uchicago.edu/Faculty_and_Research/ > > http://www.rsc.org/shop/books/2008/9780854042722.asp > > ________________________________________ > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Robbie Joosten > [robbie_joos...@hotmail.com] > Sent: Monday, April 21, 2014 4:40 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] coot problems to decrease R FREE > > Dear Peter, > >> I'm a novice of coot and ccp4. Now I'm doing refinement using both refmac5 >> and coot.Here are some problems I'm facing. Really hope you can give me >> some suggestions. >> >> 1、THE RESOLUTION OF THE DATA IS 2.5 angstrom. After first refinement of >> refmac5 I got R factor which is 0.26 and R FREE which is 0.31. My question > is >> what the final R factor and R FREE should be after several rounds of >> refinement by refmac5 and coot. > As low as it can reasonably be. Which is of course a lousy target but the > purpose of model refinement is to make it as good as possible, not to > achieve a certain R-free. That said, at this resolution, the PDB average is > about 25% for R-free. > >> 2、At which map level（e/A3 or rmsd）should I refine the data by coot? > You refine against the whole map, but for viewing purposes you should change > the map contour level during building. For the majority of the map you > should be able to see stuff well above an rmsd of 1.0 for the 2mFo-DFc map. > At the difficult bits you may need to go below 1.0, but no so low that you > can fool yourself. The proper height for the difference map depends as a bit > on the situation. I usually just turn it down until obvious noise peaks > (small negative peaks in the solvent) appear. That typically ends up > somewhere between 3 and 4 rmsd. > >> 3、Can you give me some tips and strategies about how to use coot to >> decrease R free? now I just use some basic tricks such as fit density and >> Ramachandran plot to refine the data. > This is what I do for your kind of resolution: set the weight for refinement > in COOT to 50 or 40 (the default is 60) and switch on torsion restraints and > possibly Ramachandran restraints. Then I go through the entire structure > residue by residue (space bar) refining windows of three residues (with the > 't' key) and fixing everything that I can (that includes obvious solvent > molecules). Symmetry should always be switched on and NCS ghosts (if > available too). When that is done you can focus on the remaining difficult > bits by looking at the difference map peaks in COOT and at validation > reports from WHAT_CHECK, and MolProbity. > > So now for the bit where I plug my own stuff: you can try PDB_REDO > (http://xtal.nki.nl/PDB_REDO for the server, there is also a stand-alone > version) to take a lot of work out of your hands. It optimises your > refinement in Refmac and, rebuilds side-chain and tries to find peptide > flips that improve you Ramachandran plot and fit with the maps. You also get > a lot of validation information that may help you with further rebuilding of > your model. > > Cheers, > Robbie >> >> >> Best regards, >> >> >> Peter Chen >> >
