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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: [log in to unmask] <[log in to unmask]> Sent: Thursday, February 6, 2020 5:01:14 PM To: Barone, Matthias Cc: [log in to unmask] 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" <[log in to unmask]> 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 <[log in to unmask]> on behalf of Tim Gruene <[log in to unmask]> Sent: Tuesday, February 4, 2020 9:24:24 AM To: [log in to unmask] 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- [log in to unmask] 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" <[log in to unmask]> 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 <[log in to unmask]> > Sent:Monday, February 3, 2020 7:14:25 PM > To:Barone, Matthias > Cc:[log in to unmask] > 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 <[log in to unmask]> > 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