Welcome to ROSIE Rosetta Online Server that Includes Everyone

Overview:

The Rosetta-Vienna RNP-ddG method is used to calculate relative binding affinities for RNA-protein complexes. The method involves two main steps: first, relaxation of the starting structure and second, calculation of the relative binding affinities. The relaxation step should not be skipped, but if you have previously run ddG calculations using this method, and you have the relaxed PDBs that were generated, then you can input these and opt to skip the relaxation step. If you have never run this method or you don't have the final PDBs from the relaxation step, then you should not skip this step.

Required input files:

1. A structure of an RNA-protein complex in PDB format. This structure should only contain canonical RNA and protein residues, with RNA residues specified as A, U, G, and C. The PDB should not contain HETATMs. RNA and protein should be separate chains.


2. A list of sequences for which to calculate binding affinities relative to the sequence found in the starting structure. This file should also include the free energies of the RNA as computed by Vienna RNAfold. One sequence and the corresponding RNAfold energy should be specified per line. The sequences can either be the full sequence of the complex (RNA and protein), or just the RNA sequence. If the protein sequence is not specified, then no mutations to the protein will be made. An example of the contents of a sequence file is shown below:
   
ugaggcucaccca -1.62
ugaggagcaccca -1.86

Here, -1.62 (kcal/mol) is the ensemble free energy of the sequence ugaggcucaccca (this can be calculated at the command line with RNAfold -p, or can be calculated using the Vienna RNAfold webserver). The energies shown above were calculated at 25C. The lengths of the sequences in the sequence file must all be the same and must be the same as the length of the sequence in the input PDB file (for the sequence file shown above, the RNA in the input PDB file must be 13 nucleotides). Only 100 sequences allowed per run.

Important notes:

• This webserver only uses the low-res method for calculating relative binding affinites. This means that the RNA and protein backbones will remain fixed -- this is not recommending for calculating relative binding affinities for RNA mutations that disrupt base pairs (i.e. a mutation that changes a G-C base pair to G-G).
• The score function used here is rnp_ddg.wts.
• This method does not consider shifts in the register of RNA-protein binding.
• This webserver only allows calculations for 100 sequences in a single run.
• The default (and recommended) number of times the starting structure should be relaxed before carrying out ddG calculations is 100. The 20 best scoring structures will be used to make ddG calculations, and results will be averaged. If this number is set to less than 20, then only that number of structures will be used to make ddG calculations.
• More documentation for the Rosetta-Vienna RNP-ddG method is available here.

We welcome scientific and technical comments on our server. For support please contact us at Rosetta Forums with any comments, questions or concerns.

Modeling tools developed by the Das Lab at Stanford University. The Rosie implementation was developed by Kalli Kappel.