Using ab initio models¶

UNIX (Linux|Mac)¶

$CCP4/bin/ample \
-fasta input/toxd_.fasta \
-models  ../../testfiles/decoys.tar.gz \
-mtz input/1dtx.mtz \
-nmodels 30 \
-percent 50 \
-num_clusters 1 \
-use_shelxe True \
-nproc 5 \
-show_gui True \

Windows¶

%CCP4%\bin\ample.bat ^
-fasta input\toxd_.fasta ^
-mtz input\1dtx.mtz ^
-percent 50 ^
-quick_mode True ^
-nproc 6 ^
-models ..\..\tests\testfiles\models ^
-show_gui True

First we set the path to the location where ROSETTA is installed. This is then input into ample using the -rosetta_dir flag.

Next we need to provide the locations of our input files, this is done using the following flags:

• -fasta_input – location of the FASTA file.
• -mtz_input – location of the MTZ file.

UNIX (Linux|Mac) only:

• -frags_3mers – location of the 3 residue fragment from the Robetta server.
• -frags_9mers – location of the 9 residue fragment from the Robetta server.
• -nmodels – (optional, default 1000) flag to specify the number of models we want to make with ROSETTA.

Finally we can specify some options about how AMPLE will run. Here we use:

• -percent – flag which specifies by what percentage to truncate the models.
• -use_shelxe – specifies whether we should use shelxe or not.
• -nproc – lets you specify how many processors you want to use.

For a full list possible options see AMPLE options.

Ensembling Results¶

There is a brief summary of the type of truncation that was undertaken and then a table listing each ensemble. The columns of the table are:

• Name: the name of the ensemble. This is used to name the pdb file and the directory where mrbump carries out molecular replacement.
• Truncation Level: the percentage of the model remaining after the varying residues were pruned away.
• Variance Threshold: AMPLE constructs ensembles by pruning back the most variable residues based on their variance as calculated by THESEUS. The variance threshold is the THESEUS variance score for the most variable residue that remains in this ensemble.
• No. Residues: the number of residues for each model in the ensemble.
• Radius Threshold: the truncated models are sub-clustered after truncation under 3 different radius thresholds to create the ensemble, and this is the radius used for this sub-cluster.
• No. Decoys: the number of models within this ensemble.
• Number of Atoms: the number of atoms for each model in the ensemble.
• Sidechain Treatment:
  • allatom – all sidechains were retained
  • reliable – MET, ASP, PRO, GLN, LYS, ARG, GLU, SER were retained
  • polyAla – all sidechains were stripped back to polyalanine

MrBUMP Results¶

This section displays a table with the results of running MrBUMP on each of the ensembles, for this example you will have information for the following headings.

• ensemble_name: this matches the name from the ensemble section.
• MR_program: the program used for Molecular Replacement.
• Solution type: the MrBUMP categorisation of the solution
  • GOOD - final Rfree <=0.35
  • MARGINAL - final Rfree <= 0.48 OR final Rfree <= 0.5 and the ratio between the initial and final Rfree is <= 0.8, OR final Rfree <= 0.55 and the ratio between the initial and final Rfree is <= 0.95
  • POOR - anything else
  • no_job_directory - a script has been prepared, but the job hasn’t been run yet
  • unfinished - the job is running or has stopped without generating any results
• PHASER_LLG: the PHASER log-likelihood gain for the Molecular Replacement solution.
• PHASER_TFZ: PHASER Translation Function Z-score for the Molecular Replacement solution.
• REFMAC_Rfact: Rfact score for REFMAC refinement of the Molecular Replacement solution.
• REFMAC_Rfree: Rfree score for REFMAC refinement of the Molecular Replacement solution.
• SHELXE_CC: SHELXE Correlation Coefficient score after C-alpha trace.
• SHELXE_ACL: Average Chain Length of the fragments of the SHELXE C-alpha trace.