Content:
This is an attempt of giving an introduction to our main software components within a very short time. It only gives a taster, but should be enough to get you started. Much more introductory material is available for each program:
Because of the limited time available for this demo, sometimes non-default options will be used to speed things up. Those will be clearly indicated.
Whenever timings are given, they are for a i7-2720QM laptop with 8Gb of memory.
Some examples and tutorials are given on the autoPROC wiki.
We're going to use the data for 1O22 from the JCSG. It is
This peak dataset consists of 90 images (tm0875_8p44_1_E1_001.img to tm0875_8p44_1_E1_090.img). We can run this with some additional automation using the process command from a terminal/shell:
% cd /where/ever/images % process -M automatic -d 01 | tee 01.lis
which should give us after about 3 minutes a processed dataset (using XDS, POINTLESS and SCALA/AIMLESS as part of autoPROC) with
Overall InnerShell OuterShell --------------------------------------------------------------------------- Low resolution limit 38.229 38.229 1.958 High resolution limit 1.951 8.544 1.951 Rmerge 0.061 0.034 0.491 Ranom 0.050 0.026 0.445 Rmeas (within I+/I-) 0.059 0.030 0.545 Rmeas (all I+ & I-) 0.066 0.038 0.555 Rpim (within I+/I-) 0.031 0.016 0.307 Rpim (all I+ & I-) 0.025 0.016 0.249 Total number of observations 84852 1077 669 Total number unique 13110 207 151 Mean(I)/sd(I) 24.9 57.3 2.8 Completeness 97.4 100.0 100.0 Multiplicity 6.5 5.2 4.4 Anomalous completeness 95.4 95.7 94.0 Anomalous multiplicity 3.6 3.6 2.4
There are a few interesting warnings/notes from this data processing run. First we get
WARNING : the selected indexing solution uses less than 80% of initial spots - please check this carefully for ice-rings or minor lattices. NOTE : automatically added 10 EXCLUDE_RESOLUTION_RANGE cards - based on automatic analysis (see 01/xds_spots2res.log for details)
which is due to ice-rings - shown in image 01/SPOT.noHKL.png:
Please have a look at the examples and tutorials on the SHARP/autoSHARP wiki.
We can use the data just processed for solving the structure with autoSHARP (run through the Sushi http-interface). Apart from switching on the fast path
we're going to use all defaults:
After about 10 minutes we should have
This model has 145 out of 170 residues buit with R/Rfree = 0.219/0.259 : Remember, the deposited model contains 149 residues - so we are basically finishing with a complete model.
Please make sure to have a BUSTER reference card handy. You should also check the various examples, tutorials and FAQs on the BUSTER wiki.
We can take the first (already very good) model and MTZ file - eden_flat_47.8pc_warpNtrace.pdb and eden_flat_47.8pc_warpNtrace.mtz - to run refinement with BUSTER on it. There are a few things we might want to remember at this stage:
First create a corrected PDB file with
% met2mse.sh eden_flat_47.8pc_warpNtrace.pdb
to give us eden_flat_47.8pc_warpNtrace_MSE.pdb:
Then let's first run a simple map-calculation with BUSTER ussing the refine command from a terminal/shell:
% refine -p eden_flat_47.8pc_warpNtrace_MSE.pdb \ -m eden_flat_47.8pc_warpNtrace.mtz \ FormfactorCorrection="Se:-7" \ -M MapOnly -d BUSTER.01 | tee BUSTER.01.lis
which gives us R/Rfree = 0.225/0.261 (quite similar to the values from the ARP/wARP stage of autoSHARP).
A more complete initial refinement with BUSTER at this stage could be run with
% refine -p eden_flat_47.8pc_warpNtrace_MSE.pdb \ -m eden_flat_47.8pc_warpNtrace.mtz \ FormfactorCorrection="Se:-7" \ -M WaterUpdatePkmaps -M TLSbasic \ -d BUSTER.02 | tee BUSTER.02.lis
which results in R/Rfree = 0.199/0.244 after about 8 minutes (remember that read BUSTER refine jobs runs several so-called BIG cycles where eg. bulk solvent mask and solvent structure are updated):
How does the model geometry improve? Looking at some MolProbity scores:
___________________________________________________________________________ | |Clashscore, all atoms: |23.08 |25th percentile* (N=1784, all | |All-Atom|_________________________|______|resolutions)_____________________| |Contacts|Clashscore is the number of serious steric overlaps (> 0.4 AA) per| |________|1000_atoms._______________________________________________________| | |Poor_rotamers____________|5.93%_|Goal:_<1%________________________| | |Ramachandran_outliers____|0.00%_|Goal:_<0.2%______________________| | |Ramachandran_favored_____|98.62%|Goal:_>98%_______________________| |Protein |Cb_deviations_>0.25AA____|1_____|Goal:_0__________________________| |Geometry|MolProbity score^ |2.44 |50th percentile* (N=27675, 0AA - | | |_________________________|______|99AA)____________________________| | |Residues_with_bad_bonds:_|0.00%_|Goal:_0%_________________________| |________|Residues_with_bad_angles:|0.00%_|Goal:_<0.1%______________________|
____________________________________________________________________________ | |Clashscore, all atoms: |5.36 |96th percentile* (N=821, 1.95AA +/| |All-Atom|_________________________|______|-_0.25AA)_________________________| |Contacts|Clashscore is the number of serious steric overlaps (> 0.4 AA) per | |________|1000_atoms.________________________________________________________| | |Poor_rotamers____________|4.44%_|Goal:_<1%_________________________| | |Ramachandran_outliers____|0.00%_|Goal:_<0.2%_______________________| | |Ramachandran_favored_____|99.31%|Goal:_>98%________________________| |Protein |Cb_deviations_>0.25AA____|1_____|Goal:_0___________________________| |Geometry|MolProbity score^ |1.78 |85th percentile* (N=13349, 1.95AA | | |_________________________|______|+/-_0.25AA)_______________________| | |Residues_with_bad_bonds:_|0.00%_|Goal:_0%__________________________| |________|Residues_with_bad_angles:|0.00%_|Goal:_<0.1%_______________________|
We can now checking the structure against the density (plus geomeric problems) using
% visualise-geometry-coot BUSTER.02
After improving the model interactively, additional refinement jobs could be run either directly through the Coot interface or again on the command-line.
Remember, please also read about grade (our geometric restraints generator) and the detailed examples.