The scripts and input files that accompany this demo can be found in the demos/public directory of the Rosetta weekly releases.

KEYWORDS: NUCLEIC_ACIDS EXPERIMENTAL_DATA RNA

This demo illustrates the ERRASER (Enumerative Real-Space Refinement ASsitted by Electron density under Rosetta) protocol, which improves an RNA crystallographic model using Rosetta under the constraint of experimental electron density map. It was written in Mar. 2012, by Fang-Chieh Chou (fcchou at stanford dot edu) and based on a paper to be published:

• Chou, F.C., Sripakdeevong, P., Dibrov, S.M., Hermann, T., and Das, R. Correcting pervasive errors in RNA crystallography with Rosetta, arXiv:1110.0276.

A preprint is available at:

## Setting up the demo

The example input files are in rosetta_input; you may wish to copy them locally with the command:

cp rosetta_inputs/* ./

Python codes needed to run the job are located at rosetta/rosetta_tools/ERRASER/

The following setup steps are required prior to running this demo.

2. Ensure you have correctly setup PHENIX. As a check, run the following command:

phenix.rna_validate

3. Check if you have the latest python (v2.7) installed. If not, go to the rosetta/rosetta_tools/ERRASER/ folder and run

./convert_to_phenix.python

This will change the default python used by the code to phenix-built-in python, instead of using system python.

4. Set up the environmental variable "$ROSETTA", point it to the Rosetta folder. If you use bash, append the following lines to ~/.bashrc: ROSETTA=YOUR_ROSETTA_PATH; export ROSETTA" # Change YOUR_ROSETTA_PATH to the path in your machine! Also add the ERRASER script folder to$PATH. Here is a bash example:

PATH=\$PATH:YOUR_ROSETTA_PATH/rosetta_tools/ERRASER/" # Change YOUR_ROSETTA_PATH to the path in your machine!

Now you are ready to go!

## Running the demo

As a fast quick run, run the following command:

erraser.py -pdb 1U8D_cut.pdb -map 1U8D_cell.ccp4 -map_reso 1.95 -fixed_res A33-37 A61 A65

In this example, we specify the input pdb file, ccp4 map file and the map resolution. The -pdb and -map are required option, and -map_reso is optional but recommended (default is 2.0 if no input is given). In this example, the "1U8D_cut.pdb" file is a segment cutting from a deposited PDB file. The input pdb file should follow the standard PDB format, and no pre-processing is needed. The input map must be a CCP4 2mFo-DFc map. To avoid overfitting, Rfree reflection should be removed during the creation of the map file.

Note that we also manually fixed the position of residue 33-37, 61 and 65 in chain A, therefore we will only optimize residue 62-64. The -fixed_res argument is optional.

After the job finished successfully, you should see the output pdb file "1U8D_cut_erraser.pdb" in the current folder. A sample output is in the example_output folder for comparsion.

Note that the output file is in the standard PDB format and inherits all the ligands, metals and waters from the input pdb file (these atoms are not optimized in ERRASER). The user can then refine the output model using PHENIX or other refinement packages without any post-processing.

By inspecting the structure, you should be able to see a backbone conformation change at residue 63-64 after ERRASER.

## Arguments for erraser.py (for your reference)

#### Required:

• -pdb
Format: -pdb <input pdb>

The starting structure in standard pdb format

• -map
Format: -map

2mFo-DFc map file in CCP4 format. Rfree should be excluded.

#### Commonly used:

• -map_reso
Format: -map_reso
Default: 2.0

The resolution of the input density map. It is highly recommended to input the map resolution whenever possible for better result.

• -out_pdb
Format: -out_pdb
Default: <input pdb name>_erraser.pdb.

The user can output to other name using this option.

• -n_iterate
Format: -n_iterate
Default: 1

The number of rebuild-minimization iteration in ERRASER. The user can increase the number to achieve best performance. Usually 2-3 rounds will be enough. Alternatively, the user can also take a ERRASER-refined model as the input for a next ERRASER run to achieve mannual iteration.

• -fixed_res
Format: -fixed_res
Default:
Example: A1 A14-19 B9 B10-13 (chain ID followed by residue numbers)

This allows users ton fix selected RNA residues during ERRASER. For example, because protein and ligands are not modeled in ERRASER, we recommand to fix RNA residues that interacts strongly with these unmodeled atoms. ERRASER will automatically detect residues covalently bonded to removed atoms and hold them fixed during the rebuild, but users need to specify residues having non-covalent interaction with removed atoms mannually.

• -kept_temp_folder
Format: -kept_temp_folder
Default: False

Enable this option allows user to examine intermediate output files storing in the temp folder. The default is to remove the temp folder after job completion.

#### Other:

• -rebuild_extra_res
Format/Default: Same as -fixed_res

This allows users to specify extra residues and force ERRASER to rebuild them. ERRASER will automatically pick out incorrect residues, but the user may be able to find some particular residues that was not fixed after one ERRASER run. The user can then re-run ERRASER with -rebuild_extra_res argument, and force ERRASER to remodel these residues.

• -cutpoint_open
Format/Default: Same as -fixed_res

This allows users to specify cutpoints (where the nucleotide next to it is not connected to itself) in the starting model. Since ERRASER will detect cutpoints in the model automatically, the users usually do not need to specify this option.

• -use_existing_temp_folder
Format: -use_existing_temp_folder
Default: True

When is True, ERRASER will use any previous data stored in the existing temp folder and skip steps that has been done. Useful when the job stopped abnormally and the user try to re-run the same job. Disable it for a fresh run without using previously computed data.

• -rebuild_all
Format: -rebuild_all
Default: False

When is True, ERRASER will rebuild all the residues instead of just rebuilding errorenous ones. Residues in "-fixed_res" (see below) are still kept fixed during rebuilding. It is more time consuming but not necessary leads to better result. Standard rebuilding with more iteration cycles is usually prefered.

• -native_screen_RMSD
Format: -native_screen_RMSD
Default: 2.0

In ERRASER default rebuilding, we only samples conformations that are within 2.0 A to the starting model (which is the "native" here). The user can modify the RMSD cutoff. If the value of native_screen_RMSD is larger than 10.0, the RMSD screening will be turned off.