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: [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
> >
> >
> >
> >
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> >
> >
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>
> --
> --
> 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
>
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--
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Faculty of Chemistry
University of Vienna
Phone: +43-1-4277-70202
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