Content:
For more examples see here.
We will run BUSTER from the command-line. So open a terminal, and set up the software via
module load ccp4 module load gphl
and 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):
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 adjusted and (if requested) the solvent model (waters) is updated. Void correction happnes before the final cycle.
If in ligand detection mode (with -L flag), the analysis of residual density for potenrial ligand binding sites also happens before the last big cycle.
We need the following files for this tutorial:
Please make a directory
mkdir BUSTER cd BUSTER
and download those files above into it (ensure you ait till the download is finished!). You might need to move them from the Downloads folder after downloading, e.g.
mv ~/Downloads/5z5u* . mv ~/Downloads/96U.cif .
We can run a map-only computation using
refine -p 5z5u.pdb -m 5z5u.mtz -l 96U.cif -M MapOnly -d 00 | tee 00.lis
and look at the maps with
cd 00 coot --pdb refine.pdb --auto refine.mtz
or with
cd 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?
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 01 | tee 01.lis
and look at the maps with
cd 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 01/refine-coot-0.pdb -o 01/refine-coot-0.occ refine -p 01/refine-coot-0.pdb -m 5z5u.mtz -l 96U.cif -Gelly 01/refine-coot-0.occ -d 02 | tee 02.lis
and then look again at the model and maps with
cd 02 coot --pdb refine.pdb --auto refine.mtz
See also the examples in the manual for details.
What you need to have available:
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 -M WaterUpdatePkmaps -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