|Rosetta 3.2 Release Manual|
The density constraint uses a scoring function derived from the density correlation of a model and a density map. This term is described in [DiMaio et al, JMB 2009]; some additional reading may be found in the scoring structures with density section. This method has been shown to be particularly effective in solving structures via molecular replacement from templates in the 20-30% sequence identity range.
Template files are PDB files given by "-in:file:template_pdb". Alignment files, which describe the sequence mapping from template to target, are in "Rosetta format", which is similar to a fasta-format alignment, with a few extras fields. For example:
## 1CRB_ 2qo4.PHASER.1.pdb # hhsearch scores_from_program: 0 1.00 2 DFNGYWKMLSNENFEEYLRALDVNVALRKIANLLKPDKEIVQDGDHMIIRTLSTFRNYIMDFQVGKEFEEDLTGIDDRKCMTTVSWDGDKLQCVQKGEKEGRGWTQWIEGDELHLEMRAEGVTCKQVFKKV 0 AFSGTWQVYAQENYEEFLRAISLPEEVIKLAKDVKPVTEIQQNGSDFTITSKTPGKTVTNSFTIGKEAEIT--TMDGKKLKCIVKLDGGKLVCRTD----RFSHIQEIKAGEMVETLTVGGTTMIRKSKKI --
While multiple pairs of template alignment files are accepted, it is generally recommended to run just a single template/alignment with each job.
In addition, fragment files may also be required, depending on the options selected. Rebuilding of gaps is done by fragment insertion (as in Rosetta ab initio); thus two backbone fragment files (3-mers and 9-mers) must be given. The application for building these is included with rosetta but requires some external tools/databases. The easiest way to generate fragments is to use the Robetta server (http://robetta.bakerlab.org/fragmentsubmit.jsp). The fragment files should be built with the full-length sequence; rosetta handles remapping the fragments if not all gaps are rebuilt.
bin/mr_protocols.default.linuxgccrelease \ -database ../rosetta_database \ -MR:mode cm \ -in:file:extended_pose 1 \ -in:file:fasta 1crb.fasta \ -in:file:alignment 2qo4.ali \ -in:file:template_pdb 2qo4.PHASER.1.pdb \ -loops:frag_sizes 9 3 1 \ -loops:frag_files xx1crb_09_05.200_v1_3.gz xx1crb_03_05.200_v1_3.gz none \ -loops:random_order \ -loops:random_grow_loops_by 5 \ -loops:extended \ -loops:remodel quick_ccd \ -loops:relax relax \ -relax:default_repeats 2 \ -relax:jump_move true \ -edensity:mapreso 3.0 \ -edensity:grid_spacing 1.5 \ -edensity:mapfile 2qo4.PHASER.1_2mFo-DFc.ccp4 \ -edensity:sliding_window_wt 1.0 \ -edensity:sliding_window 5 \ -cm:aln_format grishin \ -MR:max_gaplength_to_model 8 \ -nstruct 1000 \ -ignore_unrecognized_res -overwrite
For the majority of users, the only flags that will need to be changed from job to job deal with:
(1) The input structure and alignment:
-in:file:extended_pose 1 -in:file:fasta inputs/1crb.fasta -in:file:alignment inputs/1crb_2qo4.ali -in:file:template_pdb inputs/2qo4.PHASER.1.pdb
The fasta, alignment and template PDBs (see the section "Input Files" above). The flag 'extended_pose 1' should always be given.
(2) The input density map:
-edensity:mapfile inputs/sculpt_2QO4_A.PHASER.1.map -edensity:mapreso 3.0 -edensity:grid_spacing 1.5 -edensity:sliding_window_wt 1.0 -edensity:sliding_window 5
This is how the density map and scorefunction parameters are given to Rosetta. The input map (-edensity:mapfile) is CCP4 format. The flags 'mapreso' and 'grid_spacing' define the resolution of the calculated density and the grid spacing on which correlations are computed (these do not match the input file's grid but rather the grid on which Rosetta computes things). Generally I don't go lower than these values (with grid-spacing 1/2 of mapreso); for very large structures I may run on an even coarser grid to speed things up a bit.
The flag 'sliding_window_wt' is the weight on the fit-to-density term. 1.0 is generally fine. If the experimental data is low resolution (3A or worse), than you might want to drop this to 0.5. 'sliding_window' is the residue-width over which local correlations are computed. 5 is generally fine.
See the tutorial in demo/electron_density/molecular_replacement -- in particular, step 3 -- for information on creating a map from an initial molecular replacement hit.
(3) The input fragments
-loops:frag_sizes 9 3 1 -loops:frag_files inputs/aa1crb_09_05.200_v1_3.gz inputs/aa1crb_03_05.200_v1_3.gz none
Fragment files and sizes. 9 & 3 are the default produced by Robetta; 'none' means to automatically generate the 1-mer fragments from the 3-mers. See the section "Input files" for details on getting these fragment files.
(4) The number of output structures.
The number of models needed to effectively sample conformational space varies from model to model, depending on the number of gaps in the alignment and the length of those gaps. On a set of blind cases, most needed 1-2000 output strutures to inprove the model enough to ultimately solve the structure; in a few cases 100 models were sufficient.
Some lesser-used options that may be used by advanced users to control job length and sampling space of the run:
-relax:default_repeats 4 -MR:max_gaplength_to_model 8
The first option controls the length of the "relax". All our experiments used 4 repeats; for very large systems reducing this to 2 may speed things up a bit. The second option tells Rosetta not to rebuild gaps greater than length 'n' in the alignment. We found 8 and 10 both produce good results: if sampling is limited (e.g., you only have the resources to produce 100 output models) reducing this to 4 or 6 might be enough to solve the structure.
Finally, these options are manditory, and should be left as-is:
-relax:jump_move true -cm:aln_format grishin -ignore_unrecognized_res -loops:remodel quick_ccd -loops:relax relax -loops:random_grow_loops_by 5 -loops:random_order -loops:extended
This application uses the JD2 job-distributor; it respects all of the jd2 flags that control output.