RNA Structure Modeling

The RNA structure modeling algorithm in Rosetta is based on the assembly of short fragments from existing RNA crystal structures whose sequences match subsequences of the target RNA. The Fragment Assembly of RNA (FARNA) algorithm is a Monte Carlo process, guided by a low-resolution knowledge-based energy function. The models can then be further refined in an all-atom potential to yield more realistic structures with cleaner hydrogen bonds and fewer clashes; the resulting energies are also better at discriminating native-like conformations from non-native conformations.

Applications

Applications relevant to RNA structure modeling and design are available at mini/src/apps/public/rna

• The application for RNA de novo modeling is: rna_denovo.cc.
• The application for generating a source file for RNA torsions is: rna_database.cc
• The application for extracting RNA models from a "silent" file is: rna_extract.cc You can find the executable of the applications in mini/bin after successfully compiling.

Algorithm

• Generation Of Models
  A sample command line is the following:

  rna_denovo.macosgccrelease -fasta chunk002_1lnt_.fasta -nstruct 2 -out::file::silent test.out -cycles 1000
  -minimize_rna -database ~/rosetta3_database
  

  Here, the fasta file has the RNA name on the first line (after >), and the sequence on the second line. Valid letters are a,c,g, and u. The example fasta file is available in mini/test/integration/test/rna_denovo/.
• Extraction Of Models Into PDB Format
  The models from the above run are stored in compressed format in the file test.out, along with lines representing the score components. You can see the models in PDB format with the conversion command.

  rna_extract.macosgccrelease  -in:file:silent test.out -in:file:silent_struct_type  rna -database ~/rosetta3_database
  

  Note that the PDBs have residue types marked as rA, rC, rG, and rU. [This will eventually be fixed to allow the now standard PDB format for RNA.]
• Specification of Watson/Crick Base Pairs And Chainbreaks
  RNA motifs are typically ensconced within Watson/Crick double helices, and involve several strands. [The most conserved loop of the signal recognition particle is an example, and is included here as chunk002_1lnt_RNA.pdb.] You can specify the bounding Watson/Crick base pairs in a "params file" with lines like the following:

  CUTPOINT_OPEN 6    [means that one chain ends at residue 6]
  STEM PAIR 1 12 W W A    [means that residues 1 and 12 should form a base pair with their Watson-Crick edges in
  an antiparallel orientation]
  

  and then run:

  rna_denovo.macosgccrelease -fasta chunk002_1lnt_.fasta -native chunk002_1lnt_RNA.pdb -params_file chunk002_1lnt_.prm -nstruct 2
  -out::file::silent chunk002_1lnt.out -cycles 1000 -minimize_rna -database ~/rosetta3_database
  

  This command line also includes the "native" pdb, and will result in heavy-atom rmsd scores being calculated. Note again that the native pdb should have residues marked rA, rC, rG, and rU.
• Use Of Alternative Fragment Sources By default the RNA fragment assembly makes use of bond torsions derived from the large ribosome subunit crystal structure 1jj2, which have been pre-extracted in 1jj2. torsions (available in the database). If you want to use torsions drawn from a separate PDB (or set of PDBs), the following command will do the job.

  rna_database.macosgccrelease  -vall_torsions -s my_new_RNA1.pdb my_new_RNA2.pdb -o my_new_set.torsions
  -database ~/rosetta3_database
  

  The resulting file is just a text file with the RNA's torsion angles listed for each residue. Then, when creating models, use the following flag with the rna_denovo application:

  -vall_torsions my_new_set.torsions
  

  Similarly, the database of base pair geometries can be created with rna_database -jump_library, and then specified in the rna_denovo application with -jump_library_file.

Input Files

You need one input files to run RNA Strcuture Modeling.

• The fasta file: it is a sequence file for your rna.
  Optional addictional file:
• A parameter file. This can specify base pairs that are held together during the run, as well as boundaries between independent chains (see above).
• Native pdb file, if all-heavy-atom rmsd's are desired.

Options used in RNA Structure Modeling

• Sample command:

  rna_denovo.linuxgccrelease @flags > rna_denovo.log 

• Options

  -in:database                                     Path to rosetta databases. [PathVector]
  -in::fasta                                       Fasta-formatted sequence file. [FileVector]
  -out::file::silent                               Name of output file [scores and torsions, compressed format]. default="default.out" [String]
  -params_file                                     RNA params file name.[String]. For Example: -params_file chunk002_1lnt_.prm
  -in::native                                      Native PDB filename. [File].
  -out::nstruct                                    Number of models to make. default: 1. [Integer]
  -cycles                                          Number of Monte Carlo cycles.[default 1000]. [Integer]
  -minimize_rna                                    High resolution optimize RNA after fragment assembly.[Boolean]
  -filter_lores_base_pairs                         Filter for models that satisfy structure parameters. [Boolean]
  -output_lores_silent_file                        If high resolution minimizing, output intermediate low resolution models. [Boolean]
  -dump                                            Generate pdb output. [Boolean]
  -vall_torsions 			                             Source of RNA fragments. [default: 1jj2.torsions]. [Boolean]
  -jump_library_file 		                           Source of base-pair rigid body transformations if base pairs are specified.
                                                   [default: 1jj2_RNA_jump_library.dat] [String]
  

Result Interpretation

***Energy interpreter for low resolution silent output:
score     		                                   Final total score
rna_rg                                           Radius of gyration for RNA
rna_vdw                                          Low resolution clash check for RNA
rna_base_backbone                                Bases to 2'-OH, phosphates, etc.
rna_backbone_backbone                            2'-OH to 2'-OH, phosphates, etc.
rna_repulsive                                    Mainly phosphate-phosphate repulsion
rna_base_pair_pairwise                           Base-base interactions (Watson-Crick and non-Watson-Crick)
rna_base_pair                                    Base-base interactions (Watson-Crick and non-Watson-Crick)
rna_base_axis                                    Force base normals to be parallel
rna_base_stagger    				 Force base pairs to be in same plane
rna_base_stack                                   Stacking interactions
rna_base_stack_axis                              Stacking interactions should involve parallel bases.
atom_pair_constraint                             Harmonic constraints between atoms involved in Watson-Crick base
                                                 pairs specified by the user in the params file
rms                                              all-heavy-atom RMSD to the native structure

***Energy interpreter for fullatom silent output:
score                                            Final total score
fa_atr                                           Lennard-jones attractive between atoms in different residues
fa_rep                                           Lennard-jones repulsive between atoms in different residues
fa_intra_rep                                     Lennard-jones repulsive between atoms in the same residue
lk_nonpolar                                      Lazaridis-karplus solvation energy, over nonpolar atoms
hack_elec_rna_phos_phos                          Simple electrostatic repulsion term between phosphates
hbond_sr_bb_sc                                   Backbone-sidechain hbonds close in primary sequence
hbond_lr_bb_sc                                   Backbone-sidechain hbonds distant in primary sequence
hbond_sc                                         Sidechain-sidechain hydrogen bond energy
ch_bond                                          Carbon hydrogen bonds
geom_sol                                         Geometric Solvation energy for polar atoms
rna_torsion                                      RNA torsional potential.
atom_pair_constraint                             Harmonic constraints between atoms involved in Watson-Crick base pairs
                                                 specified by the user in the params file
angle_constraint                                 (not in use)
rms                                              all-heavy-atom RMSD to the native structure

***If you use the sample flag files, there are also three pdb file generated.
start.pdb:                                       Idealized, fully extended starting structure.
random.pdb:                                      Structure with randomized torsion angles.
S_0001.pdb:                                      Output of the rna denovo design.

References

1. Das, R. and Baker, D. (2007), "Automated de novo prediction of native-like RNA tertiary structures", PNAS 104: 14664-14669.
2. Das, R., Karanicolas, J., and Baker, D. (2008), "A high resolution force field for predicting and designing RNA noncanonical structure", submitted.

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