Dear Matthias,
some developers introduce new features of their refinement programs with the words " ... which has been there in SHELXL since the beginning of time". If you are only looking for two conformations, you are looking for the combination of free variable number N with part N and part -N. In case you deal with more than two conformations, take a look at SUMP (as Jon suggested). The use of free variables is easier to explain right at the computer, so please ask a colleague near you office, who is familiar with SHELXL for the details. Best, Tim On Thursday, February 6, 2020 8:10:01 PM CET Barone, Matthias wrote: > Sorry if the mail was not clear. I figured that out now yes. As I wrote in > the update, I found this stupid error I made and now everything looks good. > > Now that I got the feeling of how shelxl works, I miss one of it's features > in the pdb format, namely the possibility to link occupancies of a double > confirmation to another moiety, say a water or a double confirmation of the > ligand. It's there a way to use something similar like FVAR in a pdb file? > > > > > Dr. Matthias Barone > > AG Kuehne, Rational Drug Design > > Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) > Robert-Rössle-Strasse 10 > 13125 Berlin > > Germany > Phone: +49 (0)30 94793-284 > > ________________________________ > From: [email protected] <[email protected]> > Sent: Thursday, February 6, 2020 5:01:14 PM > To: Barone, Matthias > Cc: [email protected] > Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl > > > Hello, hope I can help. > > > OK, so here is the disp table... > > SFAC C H CL N O > > DISP $C 0.00510 0.00239 15.73708 > > DISP $H -0.00002 0.00000 0.66954 > > DISP $CL 0.18845 0.21747 1035.16450 > > DISP $N 0.00954 0.00480 28.16118 > > DISP $O 0.01605 0.00875 47.79242 > > > If we take these coordinates... > > N 3 0.414964 -0.147635 0.116896 11.00000 0.19533 > 0.44341 = > > H0A 2 0.427823 -0.138656 0.123256 11.00000 -1.50000 > > C 1 0.348035 -0.160776 0.110979 11.00000 0.20723 > 0.28451 = > > O 4 0.363785 -0.174154 0.102906 11.00000 0.21226 > 0.22954 = > > SG 5 0.177303 0.101267 0.040572 10.04000 0.06849 > 0.03024 = > > O 4 0.241304 0.071735 0.038567 10.96000 0.14982 > 0.12755 = > > ... the first N (followed by 3) is being assigned the scattering factors of > chlorine because this element is 3rd in the SFAC list. The SG (followed by > 5) is being assigned the scattering factors of O because the latter is 5th > in the SFAC list. > > I think you need to check these assignments and the chlorine occupancy are > Ok. > > Jon Cooper > > On 6 Feb 2020 11:13, "Barone, Matthias" <[email protected]> wrote: > > Dear community > here is an update of my shelxl problem. I solved it after an epiphany last > night in bed... I tried countless things to get the postive density on the > Cl under control. Markus suggested that the density came from a radiolysed > chloride, so I tried to superimpose chlorinated and radiolysed ligands. > However that did not lead to anything fruitful. > > Remember that I tried to incorporate DISP of Cl into the .ins file: > This is the original of the protein .ins, chloride just pasted as last > element: SFAC C H N O S CL > DISP $C 0.00510 0.00239 15.73708 > DISP $H -0.00002 0.00000 0.66954 > DISP $N 0.00954 0.00480 28.16118 > DISP $O 0.01605 0.00875 47.79242 > DISP $S 0.15995 0.16998 812.87489 > DISP $CL 0.18845 0.21747 1035.16450 > > The upper list only creates postive density on the Chloride, the rest of the > map is clean and looks the same as if you would omit the DISP line of Cl > alltogether. The following list is coming from the .ins file of the > converted prodrg file: > > SFAC C H CL N O > DISP $C 0.00510 0.00239 15.73708 > DISP $H -0.00002 0.00000 0.66954 > DISP $CL 0.18845 0.21747 1035.16450 > DISP $N 0.00954 0.00480 28.16118 > DISP $O 0.01605 0.00875 47.79242 > UNIT 38 48 1 5 7 > > Pasting CL as third element in the .ins file, however, created these weird > difference signals on the backbone O and N that I mentioned. You can > probably see where this is going. Here are some atoms of the protein in the > .ins file: > > N 3 0.414964 -0.147635 0.116896 11.00000 0.19533 > 0.44341 = H0A 2 0.427823 -0.138656 0.123256 11.00000 > -1.50000 C 1 0.348035 -0.160776 0.110979 11.00000 0.20723 > 0.28451 = O 4 0.363785 -0.174154 0.102906 11.00000 > 0.21226 0.22954 = SG 5 0.177303 0.101267 0.040572 > 10.04000 0.06849 0.03024 = O 4 0.241304 0.071735 > 0.038567 10.96000 0.14982 0.12755 = > > And here are some atoms of the inhibitor: > > OBM 5 0.325170 0.441790 0.181777 11.00000 0.42576 > 0.30731 = <- oxygen CE1 1 -0.036497 0.262177 0.187030 > 11.00000 0.12056 0.22455 = <- carbon HE1 2 -0.028898 0.247344 > 0.187663 11.00000 -1.20000 <- proton NAY 4 0.107745 > 0.387704 0.210972 11.00000 0.16719 0.14264 = <- nitrogen CLAA > 3 0.028744999 0.271200001 0.199305996 0.500000000 <- Chloride > > Turned out that Jon had a good feeling about the swapping of the lines and I > did not understand Tim's comment "The scattering factor is derived from the > number next to the name." Once I adjusted the numbers in the second column > of my inhibitors to match the DISP list numbering, Rfree dropped to 16.96% > and the map looks notably better (see attached snap shot). > > > Again, thank you very much for such an incredible feedback. > > Best, Matthias > > > > > > Dr. Matthias Barone > > AG Kuehne, Rational Drug Design > > Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) > Robert-Rössle-Strasse 10 > 13125 Berlin > > Germany > Phone: +49 (0)30 94793-284 > > ________________________________ > From: CCP4 bulletin board <[email protected]> on behalf of Tim Gruene > <[email protected]> Sent: Tuesday, February 4, 2020 9:24:24 AM > To: [email protected] > Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl > > Dear Jon, > > in SHELX(L), you can name your atoms foo and bar, or jon and doe, if you > like. The scattering factor is derived from the number next to the name. > The name is just that, and identifier. > > Best, > Tim > > On Monday, February 3, 2020 9:20:03 PM CET 00000c2488af9525-dmarc- > > [email protected] wrote: > > Remembered earlier that if the "CL" is not shifted one place to the left, > > Shelx and probably most other programs treat it as carbon, i.e. its > > assumed > > to have 6 rather than 17 electrons. Trust occupancies OK, too ;-? > > > > > > Jon Cooper > > > > > > On 3 Feb 2020 18:26, "Barone, Matthias" <[email protected]> wrote: > > > > > > Hi Pavel > > > > glad you write me. I was hoping you would read my post. > > > > - Yes, protons are added, both on the protein as well as on the molecule > > > > - I initially only refined protein and ligand anisotropically, now Im > > running a refinement with all atoms anisotrp except Hs. This would then > > also be the same as shelxl is doing. > > > > - Alternate conformations are modeled, also on the ligand. There are > > plenty, sure, but I think I got most of them. > > > > - I already used Water update during refine, there are some NO3s in the > > structure. I got them in. There is a second ligand somewhere as artifact. > > its density is not well defined, so I hope to get that in once the map > > clears up more. > > > > - I let phenix.refine optimize adp and chemisty weights, but as Petri > > suggested, Im manually increasing the scale factors to match the ones from > > shelxl (just to compare them properly). Im aiming for an rsmd of > > 0.02-0.03A > > like Petri suggested and keep an eye on how tight the structure is refined > > in shelxl. > > > > > > > > > > About the Rfact and the gap. Yes, thats what I was expecting. I hope if I > > add more anisotropic B fact, the Rfacts should go down to at least what > > shelxl yielded. > > > > > > > > > > thank you all again for the massive feedback, ideas and help. > > > > > > > > > > > > > > > > > > > > > > Dr. Matthias Barone > > > > AG Kuehne, Rational Drug Design > > > > > > Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) > > Robert-Rössle-Strasse 10 > > 13125 Berlin > > > > Germany > > Phone: +49 (0)30 94793-284 > > > > > > From:Pavel Afonine <[email protected]> > > Sent:Monday, February 3, 2020 7:14:25 PM > > To:Barone, Matthias > > Cc:[email protected] > > Subject:Re: [ccp4bb] refinement of 0.73A data in shelxl > > > > Hi Matthias, > > > > > > did you use correct model parameterization and optimal refinement strategy > > for the resolution? Such as: - Add H atoms; > > - Refine all but H atoms with anisotropic ADPs; > > - Model alternative conformations (that one'd expect many at this > > resolution); - Add solvent (water, crystallization cocktail components if > > you see any); - Relax restraints on geometry and ADPs; > > .... long list! > > > > > > If not, then what you have in terms of R factors is more or less what I'd > > expect. > > > > > > In the absence of obvious data pathologies, I'd expect Rwork/Rfree in > > 10-15% range, and the Rfree-Rwork gap around 1-2% or less. > > > > > > Since you mentioned Phenix refinement, I am happy to help you with details > > etc off-list. > > > > > > Pavel > > > > > > On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias <[email protected]> > > wrote: > > > > > > Dear ccp4 community > > > > Im having some problems solving a 0.73A structure. Spacegroup seems to be > > correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer > > shell CC1/2 24% and 90.4% complete. > > > > The model is nearly fully built, there is no remaining unmodelled areas. > > However, Rfactisstuck 27% in phenix, with a very distinct artifact in the > > electron map (see phenix.jpg). You can see difference density on various > > well defined sidechain atoms. Notably, they seem to follow a pattern: > > Nearly all Val CG have difference signal, as well as many backbone > > NH. Hence, I suspected that it might be a problem with the SF, since we > > recorded the DS at 0.86A. > > > > > > > > > > Hence I gave shelxl a shot: > > > > I used the refined model from phenix, converted it via pdb2ins and pasted > > the restraints created by prodrg. > > > > The shelxl hkl was produced by xdsconv, using the freeR flagging of the > > mtz > > used by phenix (no merge, friedel false). > > > > Interestingly, shelxl can bring Rfree down to 16% and almost all of > > the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg). > > Except one: the inhibitor contains a chlorinated phenylring (pdb ligand > > 2L5) which now shows massive difference density for Cl. > > > > I therefore suggested that I might deal with a wrong SF for Cl. Funny > > enough, pdb2ins does not produce a DISP line for Cl if converting the pdb > > that contains the inhibitor. Hence, I used pdb2ins and the pdb from PRODRG > > to produce SFAC for the inhibitor Cloride. I then pasted this line > > > > > > DISP $CL 0.18845 0.21747 1035.16450 > > > > > > > > into the .res file and updated the UNIT line. Shelxl runs through, and the > > density looks ok on the Chloride now. However Rfree is back up at 24% and > > the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg): now, > > very distincitvly, backbone carbonyls and NHs show difference density. > > > > Am I right in my assumption, that the SFAC of Cloride is not properly > > calculated at the given wavelenght? And if so, how do I guess it > > correctly? > > > > > > > > > > > > Thank you very much for your help! > > > > Best, matthias > > > > > > > > > > > > > > > > > > > > > > > > > > Dr. Matthias Barone > > > > AG Kuehne, Rational Drug Design > > > > > > Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) > > Robert-Rössle-Strasse 10 > > 13125 Berlin > > > > Germany > > Phone: +49 (0)30 94793-284 > > > > > > > > > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > > > > > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > > > > > > > > > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > -- > -- > Tim Gruene > Head of the Centre for X-ray Structure Analysis > Faculty of Chemistry > University of Vienna > > Phone: +43-1-4277-70202 > > GPG Key ID = A46BEE1A > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ________________________________ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 -- -- Tim Gruene Head of the Centre for X-ray Structure Analysis Faculty of Chemistry University of Vienna Phone: +43-1-4277-70202 GPG Key ID = A46BEE1A ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
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