The following is a protocol to walk you through the steps required to run a Rosetta refinement of an atomic model in a 2.2 Angstrom Beta-Gal structure.

Getting setup

Perform multi-sequence alignment with HHpred

1. Navigate to

2. Paste Beta-Gal's FASTA protein sequence into the prompt:


3. Click Submit.

4. Wait for job to finish - can take a few hours.

  • Beta-Gal took ~15 minutes.

5. Download HHR file from Results output page by clicking on the 'HHR' menu item:


6. Edit .hrr file to include only top 5 hits (or at least 3). For Beta-Gal, we used 5.

Use HHR file to create PDB files for initial docking

Now we will submit the HHR file to Rosetta on AWS to create PDB files with the correct amino acid sequence for Beta-Gal:

$ /path/to/cryoem-cloud-tools/rosetta/ --fasta=betagal.fasta --hhr=hhpred_5556431_editedTop5.hhr  --AMI=[AMI ID]

When running, you will see the following output to your terminal:

Formatting input .hrr and .fasta files to create PDB files for docking into density...

...booting up instance to format input files...

...running Rosetta file preparation on t2.micro instance...

...finished with file preparation, shutting down instance

Please dock each of these PDB files into your density (e.g. using UCSF Chimera) and then save into a text file to be provided as --pdb_list for Rosetta-CM or Rosetta-relax:






Dock PDB files into density

Now that you have formatted the PDB files with Rosetta, you will dock each of the output models (5 in total for this example) into the cryo-EM density.

To do this with UCSF Chimera, first prepare the 3D volume:

  1. Open cryo-EM density in UCSF Chimera
  2. Change 'Step' to '1' on the Volume Viewer menu
  3. Place threshold value at 0.0243 in Volume Viewer Window

Then, leaving Chimera open, for each PDB file:

  1. Open PDB file (e.g. 2017-09-28-133115-Rosetta-CM/1f4aC_201.pdb)
  2. Make just the PDB file active: Favorites > Model Panel > [Select 1f4aC_201.pdb from list] > Click 'activate only'
  3. Using mouse, roughly position into density
  4. Dock into map: Tools > Volume Data > Fit in map; Select PDB file and 3D model in menu and click 'Dock'.
  5. To check that the docking looks ok, activate all models: Favorites > Model Panel > Click 'activate all', and then rotate docked coordinate into map.
  6. This should orient the model correctly. If not, click 'Undo' and then manually reorient the PDB model as the starting position must have been too far away from the correct position.

When satisfied, save the docked coordinates:

  1. Go to: File > Save PDB
  2. Saving options:
    • Make sure that only your PDB model is selected in menu
    • Do not select 'Save displayed atoms only' OR 'Save selected atoms only'
    • Select 'Save relative to model' and make sure that the correct volume is selected.
    • Enter new file name. (Hint: Name with extension '_docked.pdb' to help keep files organized).
  3. Click 'Save'
  4. Repeat for all output PDB files.

Example saving screen:

Link to download output PDB files and docked PDB files.