Hi Careina Since my name came up I felt compelled to comment, though I don't have a definitive answer. Over-restraining of B factors is certainly a possibility and could well explain otherwise inexplicable difference density. IMO B factors are generally over-restrained. They are a bit like the R factors: tight restraints keep the B factors low and people (referees in particular!) seem to feel happier when they see low values, even though that is not necessarily optimal (this mind-set is of course known as "wishful thinking" - see Wikipedia article).
Personally I never restrain 1-3 B factors, and only use the 1-2 restraints, and even then I relax those from the default values. The 1-3 B restraints are clearly not independent of the 1-2 ones, and restraints should ideally be independent of each other, otherwise you are applying them more than once, IMO the overall B factor restraint weight should be optimised in the same way that the X-ray weight should be, by minimising Rfree or -LLfree. X-plor, CNS (and I would guess phenix.refine) have had this option for many years (optimize_rweight.inp in CNS), and it seems an excellent thing to do, Disorder is also an obvious possibility as Anthony said, and I would follow his sensible advice. Cheers -- Ian Cheers -- Ian On 23 November 2011 10:58, Eleanor Dodson <c...@ysbl.york.ac.uk> wrote: > I wish I could answer this! > One possibility is that the side-chain B values are too tightly restrained - > Ian Tickle recommends releasing these somewhat.. > > Here are the default refmac values. > THERMAL FACTORS > Weight= 1.00 > Main chain bond (1-2 neighbour) 1.5A**2 > Main chain angle (1-3 neighbour) 2.0A**2 > Side chain bond 3.0A**2 > Side chain angle 4.5A**2 > > and you could change them say to: > > > Under GUI - check geometric parameters and alter the Bfactor values > > data line becomes: temp 1.0 1.5 2.0 4.0 6.0 > > The trouble is that resyraints for ARG or MET say should be looser than > those for SER or VAL. > > But I often finish up with some inexplicable red blobs - sometimes floating > in space.. > Eleanor > > > On 11/23/2011 08:39 AM, DUFF, Anthony wrote: >> >> >> Delete (set occupancies to zero) the side chain back to CA. Do a few >> rounds of refinement and calculate Fo-Fc and examine. >> >> >> > > >> It is possible that the side chain is disordered, or ordered in multiple >> conformations. Compare the density for alternate confonformers against the >> density for CA. >> >> >> >> Alternatively, your side chain B-factor restaints might be too tight. >> >> >> >> Anthony >> >> >> >> >> >> ________________________________ >> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Careina >> Edgooms [careinaedgo...@yahoo.com] >> Sent: Wednesday, 23 November 2011 6:54 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] negative density in difference map >> >> Good morning CCP4 members >> >> I have a question about a 2F0-Fc difference map that I calculated with >> Refmac. In some instances it gives me negative (red) density around part of >> a side chain and no positive density in sight. Furthermore the entire >> residue fits well into the blue density of the complete map, including the >> part with negative density. >> I am struggling to interpret this. Does the fact that it fits the blue >> density mean that the side chain is in the correct place or does the red >> blob on part of the side chain (eg on the sulphur in a Met residue) mean >> that something funky is happening with this side chain? >> >> Thanks for any assistance >> Careina >> >
