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
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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
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