Content:
5Z5U:
7XJ5:
Looking at ligands (and all that):
We will run BUSTER from the command-line. So open a terminal on one of the processing machines and run
gphl
This will create a tutorial directly you can then work in. If you want to use the latest version of our software (to be released in the next couple of weeks), please type
gphllatest
You should now be able to see the online help via
refine -h
Note: It might be useful to make the terminal window larger, especially wide enough to avoid ugly line breaks that make it difficult to read the content of that help message.
There are some so-called macros available (collections of parameter settings for a particular task): for details see
refine -M list
As you can see, there are a lot of different flags to use, but the most common ones will be
-p <PDB> | specifies input model PDB file |
-m <MTZ> | specifies input reflection MTZ file (e.g. "staraniso_alldata-unique.mtz" or "truncate-unique.mtz" from autoPROC |
-l <CIF> | extra restraints directory for non-standard ligand/compound; several -l flags can be given |
-autoncs | to switch on use of LSSR restraints for non-crystallographic symmetry (NCS); remember that this flag is neutral if you don't have NCS |
-RB | start with a simplified rigid-body refinement cycle |
-L | if looking for a potential ligand |
Other tools you might want to look at (all will give help messages if run with -h flag):
aB_hydrogenate -full -p input.pdb -o output.pdb
Remember that one BUSTER job (i.e. what happens when running the refine command) consists of multiple so-called "big" cycles - usually 5 - with (by default) up to 100 "small" cycles (iterations) each. Between each big cycle, the bulk solvent model is updated, X-ray weight automatically adjusted and (if requested) the solvent model (waters) is updated. Void correction happens before the final cycle.
If in ligand detection mode (with -L flag), the analysis of residual density for potential ligand binding sites also happens before the last big cycle.
We need the following files for this tutorial:
The relevant files are already availabe in /data/rapidata2/SampleData2022/5z5u, so you should be able to copy them over using
cp -p /data/rapidata2/SampleData2022/5z5u/* .
But you can also fetch them directly via
wget https://www.globalphasing.com/buster/wiki/plugin/attachments/WorkshopRapiData2022/5z5u.pdb wget https://www.globalphasing.com/buster/wiki/plugin/attachments/WorkshopRapiData2022/5z5u.mtz wget https://www.globalphasing.com/buster/wiki/plugin/attachments/WorkshopRapiData2022/5z5u_all.mtz wget https://www.globalphasing.com/buster/wiki/plugin/attachments/WorkshopRapiData2022/96U.cif
For this 5Z5U example, you could also try and use the 5z5u_all.mtz file (data to 1.1 A resolution) instead of the default 1.6 A 5z5u.mtz. It is not quite clear why the structure was refined only against 1.63 A data, but intensities to 1.1A were deposited). This might be related to the severe anisotropy of the data:
Diffraction limits & principal axes of ellipsoid fitted to diffraction cut-off surface: 1.846 1.0000 0.0000 0.0000 a* 1.197 0.0000 1.0000 0.0000 b* 1.298 0.0000 0.0000 1.0000 c*
We could upload that high-resolution MTZ file (containing intensities) to the STARANISO server in order to create a new MTZ file after anisotropy analysis and correction:
PDB | STARANISO | MTZ |
5Z5T | 2TAlA08dR1vManrn | 5Z5T_staraniso.mtz |
5Z5U | sfZW76drwijLJFXi | 5Z5U_staraniso.mtz |
5Z5V | Mkca99x3EssJDrZW | 5Z5V_staraniso.mtz |
We can run a map-only computation using
refine -p 5z5u.pdb -m 5z5u.mtz -l 96U.cif -M MapOnly -d 5z5u.00 | tee 5z5u.00.lis
and look at the maps with
cd 5z5u.00 coot --pdb refine.pdb --auto refine.mtz
or with
cd 5z5u.00 visualise-geometry-coot
What can you see? Is there something interesting in the model and/or maps - especially for the ligand? Maybe some alternate conformations to be modelled? Or a wrong ligand (compare to PDB entry 5Z5T and the 96R compound, 96R.cif)?
A full refinement would look like this (see also your BUSTER reference card handout: and the manual):
refine -p 5z5u.pdb -m 5z5u.mtz -l 96U.cif -d 5z5u.01 | tee 5z5u.01.lis
and look at the maps with
cd 5z5u.01 coot --pdb refine.pdb --auto refine.mtz
or with
cd 01 visualise-geometry-coot
If you modelled some alternate conformations or partially occupied residues/atoms, you could then run
cd ../ pdb2occ -p 5z5u.01/refine-coot-0.pdb -o 5z5u.01/refine-coot-0.occ refine -p 5z5u.01/refine-coot-0.pdb -m 5z5u.mtz -l 96U.cif -Gelly 01/refine-coot-0.occ -d 5z5u.02 | tee 5z5u.02.lis
and then look again at the model and maps with
cd 5z5u.02 coot --pdb refine.pdb --auto refine.mtz
See also the examples in the manual for details.
The 7XJ5 model seems to need a bit more refinement - at least when judging from the validation report. You can take the files
or get them afresh via
fetch_PDB 7XJ5
and the ECW restraint dictionary from the Grade webserver or from here: ECW.grade_PDB_ligand.cif. You might be able to run the refinement then yourself and look at the resulting maps - especially around the ligand.
A simple map-only start would be to run
refine -p 7xj5.pdb -m 7xj5.mtz -l ECW.restraints.cif -M MapOnly -d MapOnly | tee MapOnly.lis coot 7xj5.pdb MapOnly/refine.mtz
A full refinement could be done with
refine -p 7xj5.pdb -m 7xj5.mtz -l ECW.restraints.cif -d 01 | tee 01.lis coot 01/refine 01/refine.mtz
Alternatively, you could try the automatic refinement protocol provided by aB_autorefine with
aB_autorefine -p 7xj5.pdb -m 7xj5.mtz -l ECW.restraints.cif -d 02 | tee 02.lis coot 02/refine.pdb 02/refine.mtz
which will produce a final summary - showing e.g. the progress of R/Rfree at various steps
Please note how BUSTER starts with a very similar R/Rfree as the deposited model (refined with phenix originally), but manages to significantly reduce this from 0.272/0.293 to 0.199/0.228. This might be a very beneficial (and maybe outlier) example, but it shows that sometimes the fully automatic, all-default runs can already do a fairly good job at providing a maybe better starting point for manual model corrections and interpretations.
The model here might need some re-evaluation of the metal sites (and maybe additional sites) as well as the ligand itself.
What you need to have available:
Note: If you don't have your own model/data yet and want to use an existing PDB deposition, use
fetch_PDB.sh PDBid
(with the correct PDB identifier of course) to get the model and reflection data as MTZ.
Some other tools that might be relevant for your particular problem (all support the -h flag to give you a help message):
After some initial refinement (and model building) you could also allow for automatic update of the water structure. So maybe start with something like
refine -p my.pdb -m my.mtz -l ligand.cif -autoncs -d 01 | tee 01.lis cd 01 coot --pdb refine.pdb --auto refine.mtz
(and do all necessary work in Coot, saving the coordinates at the end). The next step would then be
cd .. refine -p 01/refine-coot-0.pdb -m my.mtz -l ligand.cif -autoncs -WAT -d 02 | tee 02.lis cd 02 coot --pdb refine.pdb --auto refine.mtz
Switching on TLS refinement could be done via
refine -p 02/refine.pdb -m my.mtz -l ligand.cif -autoncs -M TLSbasic -d 03 | tee 03.lis cd 03 coot --pdb refine.pdb --auto refine.mtz
Using ADP refinement (for high resolution data):
refine -p 03/refine.pdb -m my.mtz -l ligand.cif -autoncs -M ADP -d 04 | tee 04.lis cd 04 coot --pdb refine.pdb --auto refine.mtz
Since the 20220608 release we provide another level of automation with the aB_autorefine interface to BUSTER: it combines some high-level decision making about model parametrisation with a defined sequence of individual BUSTER jobs - and might be a good starting point for initial model refinement.