Hi Clemens, > -----Original Message----- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Clemens Vonrhein > Sent: Friday, June 16, 2017 12:42 > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Problem with Mg2+ binding site refinement > > Hi, > > On Fri, Jun 16, 2017 at 10:47:20AM +0530, Prem Prakash wrote: > > Hi Mubinur, > > I got the same situation while refining my protein complex with > > Lanthanum ion complex, reducing the occupancy while refining solved the > problem. > > That might have not been the whole story through. You still want to use the > correct formfactor (as Eleanor said) for each atom in your model. The > formfactors we usually use in refinement (e.g. $CCP4/lib/data/atomsf) are > for CuKa wavelength - and the data might have been collected at a different > wavelength. Therefore those formfactors might not be correct in that case. > > For most atoms in your model that won't matter that much, since f' > doesn't change very much with resolution: > > 0.8A 0.9A 1.0A 1.1A 1.2A 1.3A 1.4A 1.5A 1.6A 1.7A > 1.8A 1.9A > 2.0A > > --------------------------------------------------------------------------------------------- > --- > C : 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.02 0.02 0.02 > 0.02 0.02 0.03 > N : 0.00 0.01 0.01 0.01 0.02 0.02 0.02 0.03 0.03 0.03 > 0.04 0.04 0.05 > O : 0.01 0.01 0.02 0.02 0.03 0.03 0.04 0.04 0.05 0.05 > 0.06 0.07 0.07 > P : 0.11 0.13 0.16 0.18 0.21 0.23 0.25 0.27 0.29 0.31 > 0.33 0.34 0.35 > S : 0.13 0.16 0.19 0.22 0.24 0.27 0.29 0.31 0.33 0.34 > 0.36 0.37 0.37 > > (S and P do have a small change though). However, for other atoms that can > be much more extreme: > > 0.8A 0.9A 1.0A 1.1A 1.2A 1.3A 1.4A 1.5A 1.6A 1.7A > 1.8A 1.9A > 2.0A > > --------------------------------------------------------------------------------------------- > --- > Br : -1.05 -3.12 -2.23 -1.62 -1.31 -1.11 -0.95 -0.82 -0.71 -0.61 > -0.52 -0.45 - > 0.37 > Cl : 0.15 0.19 0.22 0.25 0.27 0.30 0.32 0.34 0.35 0.36 > 0.37 0.37 0.37 > Ca : 0.23 0.27 0.30 0.33 0.34 0.35 0.35 0.35 0.32 0.29 > 0.25 0.19 0.11 > Mg : 0.05 0.06 0.08 0.09 0.11 0.13 0.14 0.16 0.17 0.19 > 0.20 0.22 > 0.23 > La : -0.47 -0.37 -0.34 -0.38 -0.50 -0.72 -1.04 -1.50 -2.11 -2.93 > -4.06 -5.75 - > 8.32 Se : -0.64 -1.62 -3.48 -1.96 -1.52 -1.26 -1.08 -0.94 -0.81 > -0.71 -0.62 - > 0.53 -0.46 > > (running CROSSEC will give you those numbers). > > When refining against your data at a wavelength different than CuKa, you > need to adjust your formfactors accordingly (see e.g. [1]). So if you > collected > your Lanthanum dataset at slightly longer wavelengths than CuKa, you > should tell your refinement program to adjust its scattering factors - and > since f' is lower, this might already remove that negative density without the > need to fudge it via a reduction in occupancy. Of course, you could still > have a > partially occupied ion, but with the correct formfactor you will at least have > the chance to get the occupancy adjustment right. Exactly. The occupancy column is to model the atomic occupancy, it is not a fudge factor.
> I always thought that the very common approach of adjusting SE atom > occupancy down to 0.75 or 0.80 in Se-MET (MSE) structures deposited in the > PDB is not necessarily due to only partial incorporation of Se-MET (versus S- > MET) during expression, but can sometimes be attributed to using the wrong > formfactor during refinement. Yes, we looked at that in PDB-REDO as well. Also hacking the Se occupancy to match the effective scattering is only half the story as the rest of the time there is a sulfur atom rather than nothing. > Of course, it is very helpful if data processing packages keep the wavelength > information in the MTZ file correct. Then one can at least compute > theoretical f' values for a given wavelength. Using measured f' values from a > good fluorescence scan when collecting data close to the edge is obviously > even better. Yes, please. On the PDB-REDO server we get a lot of MTZ files without wavelength and it is not obvious where that problem comes from. In our lab we mostly use Synchrotron -> XDS -> Aimless and that always neatly puts the wavelength in the MTZ file without any particular effort. Cheers, Robbie > > Cheers > > Clemens > > [1] > https://www.globalphasing.com/buster/wiki/index.cgi?AutoBusterExampleF > ormfactor > (slightly out-of=date, but gives the idea). Assuming the > wavelength in the MTZ header is correct (e.g. from autoPROC, > www.globalphasing.com/autoproc/) and you are away from the edge, > using AutomaticFormfactorCorrection=yes is all you need to add to > the command-line. Otherwise one can use > e.g. FormfactorCorrection="Se:-6.5 Ca:0.27" or such). > > > > Good luck > > P.P > > > > On Thu, Jun 15, 2017 at 11:17 PM, Pavel Afonine <pafon...@gmail.com> > wrote: > > > > > Hi Mubinur, > > > > > > try without "metal restraints" and see if that helps. As others > > > suggested, make sure 2+ is present in rightmost column of PDB file. > > > The side may be partially occupied, so refining occupancy of Mg2+ is not a > bad idea. > > > > > > Pavel > > > > > > On Wed, Jun 14, 2017 at 2:44 PM, Mohammad Rahman > > > <mohammad.rah...@uef.fi> > > > wrote: > > > > > >> Dear All, > > >> > > >> I am trying to refine a tetrameric enzyme structure that was > > >> determined at 2.7 Å. The structure contains a Mg2+ binding site in each > monomer. > > >> After refinement, in Mg2+ binding site negative density (red) has been > > >> found as in pictures. I am using Phenix refine, and during > > >> refinement, metal restrains were used. Herewith I have attached the > > >> refinement statistics. > > >> > > >> please help me to overcome this problem. > > >> > > >> Thank you > > >> > > >> -Mubinur > > >> > > >> > > -- > > *-------------------------------------------------------------- > * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com > * Global Phasing Ltd., Sheraton House, Castle Park > * Cambridge CB3 0AX, UK www.globalphasing.com > *--------------------------------------------------------------
