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Molecular simulations for METL

This repository facilitates high-throughput Rosetta runs to compute energy terms for protein variants. For more information, please see the metl repository and our manuscript:

Biophysics-based protein language models for protein engineering.
Sam Gelman, Bryce Johnson, Chase Freschlin, Sameer D'Costa, Anthony Gitter⁺, Philip A Romero⁺.
bioRxiv, 2024. doi:10.1101/2024.03.15.585128
⁺ denotes equal contribution.

Users of this workflow should cite Rosetta in addition to METL.

Table of Contents

• Setup
• Preparing PDB files for Rosetta
• Computing Rosetta energy terms for protein variants
• Running with HTCondor
  • Packaging a minimal distribution of Rosetta
  • Packaging the Python environment
  • Prepare a PDB file for use with Rosetta
  • Generating variant lists
    • Generating all possible variants
    • Generating variants using the subvariants algorithm
  • Prepare an HTCondor run
  • Processing results
    • Parse the results files
    • Add results to a database

Setup

Install Rosetta v3.13 from rosettacommons.org. See the website for instructions on acquiring a license and installing the software.

There are two Python environments in the setup directory. Install them with Anaconda.

conda env create -f setup/clean_pdb_env.yml
conda env create -f setup/metl-sim_env.yml

Installation typically takes approximately 5 minutes.

The main environment is named metl-sim.

conda activate metl-sim

The other environment, named clean_pdb, is exclusively for running Rosetta's clean_pdb.py and clean_pdb_keep_ligand.py, which require Python 2. There is no need to manually activate this environment. It will be automatically activated when preparing PDB files for use with Rosetta.

Preparing PDB files for Rosetta

Raw PDB files like those acquired from Protein Data Bank need to be prepared for use with Rosetta. Our approach is based on the recommendation in the Rosetta documentation.

1. Run Rosetta's clean_pdb.py or clean_pdb_keep_ligand.py.
2. Relax with all-heavy-atom constraints.

   -relax:constrain_relax_to_start_coords
   -relax:coord_constrain_sidechains
   -relax:ramp_constraints false
   -ex1
   -ex2
   -use_input_sc
   -flip_HNQ
   -no_optH false
   

3. Select the lowest energy structure generated in the previous step.

The main script for this pipeline is prepare.py, and it will run the above steps for you (no need to do it manually).

Example

Let's prepare 2qmt.pdb for use with Rosetta. This PDB file was downloaded from Protein Data Bank and is located at pdb_files/raw_pdb_files/2qmt.pdb.

Make sure the metl-sim conda environment is active. Then, call the following command from the root directory of this repository.

python code/prepare.py --rosetta_main_dir=<path_to_rosetta_main_dir> --pdb_fn=pdb_files/raw_pdb_files/2qmt.pdb --relax_nstruct=10 --out_dir_base=output/prepare_outputs 

Note
You need to specify the path to your Rosetta installation's main folder. It may look similar to:
/Users/<username>/rosetta_bin_mac_2021.16.61629_bundle/main

Additional arguments specified in this example:

For a full list of parameters, call python code/prepare.py -h.

Running this example can take approximately 5-10 minutes depending on CPU speed.

When you call this command, the prepare.py script will:

• Create an output directory named 2qmt under output/prepare_outputs
• Call Rosetta relax to generate 10 potential structures
• Automatically select the lowest energy structure and copy it to 2qmt_p.pdb
• Place the remaining structures and other outputs in the working_dir directory.

Computing Rosetta energy terms for protein variants

The energize.py script can be used to compute Rosetta energy terms for protein variants.

This script runs our full Rosetta pipeline, consisting of multiple steps to:

1. Introduce the variant's mutations to the base structure using a resfile [1] [2]
2. Relax the updated structure to compute the main energy terms [1] [2] [3]
3. Compute custom filter-based energy terms [1] [2]
4. Compute centroid energy terms [1]

This script calls Rosetta binaries using the subprocess library. Some hyperparameters for Rosetta can be specified as arguments to this script. This script will either pass those hyperparameters along to Rosetta as command line arguments, or it will modify the templates in the templates directory to account for the hyperparameters. Other hyperparameters are hardcoded in various files in the templates directory. For ease of use and reproducibility, arguments to this script can be stored in text files in the energize_args directory. The arguments used to generate Rosetta data for our manuscript are in condor_set_2.txt

This script processes the results from all of these steps into a single dataframe.

Example

Let's compute Rosetta energy terms for a variant of the PDB file we prepared in the previous step, 2qmt_p.pdb. The energize.py script accepts variants in a text file. I explain how to generate variant lists in the next section. For now, I created a sample text file with two variants, 2qmt_p_example.txt.

Make sure the metl-sim conda environment is active. Then, call the following command from the root directory of this repository.

python code/energize.py @energize_args/example.txt --rosetta_main_dir=<path_to_rosetta_main_dir>  --variants_fn=variant_lists/2qmt_p_example.txt --save_wd

Note we are using the @energize_args/example.txt argument to insert arguments from that file. Additionally, we are specifying the path to the Rosetta installation, the file containing the variants to run, and an additional flag --save_wd to save the working directory for each variant. For condor runs, there is no need to specify the --save_wd flag, as it will generate too much output.

Running this example takes approximately 2-3 minutes depending on CPU speed.

By default, the output will be placed in the output/energize_outputs directory. The output consists of multiple files:

• args.txt which contains the arguments used to run the script
• energies.csv which contains the computed energy terms for each variant
• hparams.csv which contains the hyperparameters used to compute the energy terms
• job.csv which contains information about the run
• If the --save_wd flag is specified, the output will also contain working directories for each variant, which contain a number of additional log files and structure files output by Rosetta.

Running with HTCondor

The main steps for setting up a metl-sim HTCondor run are:

1. Package a minimal distribution of Rosetta and upload it to SQUID or OSDF (rosetta_minimal.py)
2. Package the Python environment and upload it to SQUID or OSDF
3. Prepare a PDB file for use with Rosetta (prepare.py)
4. Generate a list of variants for which you want to compute energies (variants.py)
5. Prepare an HTCondor run (condor.py)

Packaging a minimal distribution of Rosetta

The rosetta_minimal.py script can be used to:

• Create a minimal distribution of Rosetta
• Compress it into a single .tar.gz file
• Encrypt the .tar.gz file with a password using the openssl library
• Split the encrypted .tar.gz file into multiple ~700mb files

Note
You need to package the Linux version of Rosetta for running on Linux servers like those available from CHTC and OSG

Note
If you add custom Rosetta code to compute new energy terms, you will need to modify the rosetta_minimal.py script to include your code dependencies in the minimal distribution

To create the minimal distribution of Rosetta, call the following command from the root directory of this repository.

python code/rosetta_minimal.py --gen_distribution --rosetta_main_dir=<path_to_rosetta_main_dir> --out_dir=rosetta_minimal

where <path_to_rosetta_main_dir> is the path to full Rosetta distribution. It may look similar to: /Users/<username>/rosetta_bin_linux_2021.16.61629_bundle/main. The minimal distribution will be created in the rosetta_minimal directory.

To package the Rosetta distribution for SQUID or OSDF:

python code/rosetta_minimal.py --prep_for_squid --out_dir=rosetta_minimal --squid_dir=output/squid_rosetta --encryption_password=password

The packaged distribution will be created in the output/squid_rosetta directory.

Note
You must specify an encryption password to prevent unauthorized access to the Rosetta distribution. Make sure to update pass.txt with the password you choose. The password contained in pass.txt is what will be used to decrypt Rosetta later on and must match the encryption password.

Once you have the packaged Rosetta distribution, upload it to OSDF. Then, modify osdf_rosetta_distribution.txt to list the OSDF paths to the Rosetta distribution files you uploaded. The file currently contains the following example paths:

osdf:///chtc/staging/sgelman2/squid_rosetta_2023-10-30_21-53-58/rosetta_min_enc.tar.gz.aa
osdf:///chtc/staging/sgelman2/squid_rosetta_2023-10-30_21-53-58/rosetta_min_enc.tar.gz.ab
osdf:///chtc/staging/sgelman2/squid_rosetta_2023-10-30_21-53-58/rosetta_min_enc.tar.gz.ac

Packaging the Python environment

You must package the Python environment and upload it to SQUID or OSDF. CHTC has instructions on how to do this here. To make this process easier, I created a helper script, package_env.sh.

To package the Python environment, perform the following steps:

1. Create a working directory on the submit node named environment
2. Upload htcondor/package_env.sh and setup/metl-sim_env.yml to the environment directory
3. CD into the environment directory on the submit node
4. Rename metl-sim.yml to environment.yml (this is what package_env.sh expects)
5. Run package_env.sh and wait for it to finish
6. Transfer the resulting metl-sim_env.tar.gz file to OSDF
7. Modify osdf_python_distribution.txt to list the OSDF path to the Python environment file you uploaded. See the current file contents for an example.

Prepare a PDB file for use with Rosetta

See the above section on Preparing PDB files for Rosetta.

If preparing your PDB file(s) locally is too slow, you can create an HTCondor run to do it.

Todo: add instructions for this

Generating variant lists

The variants.py script can be used to generate variant lists. It has three modes of operation: all, random, subvariants.

Generating all possible variants

For small proteins with a small number of mutations, it may be feasible to generate all possible variants. Here is how to generate all possible single amino acid substitution variants for the PDB file 2qmt_p.pdb we prepared in the previous section.

python code/variants.py all --pdb_fn=pdb_files/prepared_pdb_files/2qmt_p.pdb --num_subs_list 1

You can generate all double amino acid substitution variants by specifying --num_subs_list 2. By default, the output will be written to variant_lists/2qmt_p_all_NS-1.txt. You can specify a different output directory using the --out_dir argument.

Generating variants using the subvariants algorithm

We implemented a subvariants sampling algorithm to ensure that all possible subvariants are included in the variant list.

Given:

• The maximum and minimum number of substitutions
• The desired number of variants

The algorithm initializes an empty list of variants and performs the following until the desired number of variants is reached:

1. Generate a random variant with the maximum number of substitutions.
2. Check if the new variant is already in the list of variants. If it is, go back to step 1.
3. Add the new variant to the list of variants.
4. Generate all possible subvariants of the new variant, starting with the max number of substitutions minus one and down to the minimum number of substitutions.
5. For each subvariant, check if it is in the list, and if it is not, add it to the list.

In the previous step, we generated all possible single amino acid substitution variants for the PDB file 2qmt_p.pdb. Now, let's use the subvariants algorithm to generate 100,000 variants with a maximum of 5 substitutions and a minimum of 2 substitutions.

python code/variants.py subvariants --pdb_fn=pdb_files/prepared_pdb_files/2qmt_p.pdb --target_num 100000 --max_num_subs 5 --min_num_subs 2

By default, the output will be written to the variant_lists directory.

Prepare an HTCondor run

The condor.py script can be used to prepare an HTCondor run. You can specify arguments to this script directly on the command line, or you can specify them in arguments files stored in the run_defs directory. I prefer to create an argument file for every condor run for record-keeping and reproducibility.

Here is how you would create an argument file for a condor run to compute energy terms for the single substitution variants we generated in the previous section. Note each command line argument is specified on a separate line.

Contents of htcondor/run_defs/gb1_example_run.txt

--run_type
energize
--run_name
gb1_example_run
--energize_args_fn
energize_args/condor_set_2.txt
--master_variant_fn
variant_lists/2qmt_p_all_NS-1.txt
--variants_per_job
-1
--osdf_python_distribution
htcondor/templates/osdf_python_distribution.txt
--osdf_rosetta_distribution
htcondor/templates/osdf_rosetta_distribution.txt
--github_tag
v1.0

You can then generate the HTCondor run using the following command:

python code/condor.py @htcondor/run_defs/gb1_example_run.txt

The script will generate a run directory and place it in output/htcondor_runs. From there, you can upload the run directory to your HTCondor submit node. You can then submit the run using submit.sh, which should be located in the run directory.

Processing results

The HTCondor run will produce a log directory for each job. The log directory contains 4 files: args.txt, job.csv, hparams.csv, and energies.csv.

After the HTCondor run, transfer these log directories to your local machine for processing. I recommend compressing them before the file transfer:

tar -czf output.tar.gz output

Then untar the file on your local machine:

tar -xf output.tar.gz

Make sure to extract the output into the same condor run directory that you produced with condor.py in the Prepare an HTCondor run section.

Parse the results files

The process_run.py script can be used to parse the results files and combine them into a single dataframe.

Run it with the following command, specifying the mode stats and the main run directory of the HTCondor run:

python code/process_run.py stats --main_run_dirs output/htcondor_runs/my_condor_run

This will produce a directory named processed_run inside in the main run directory. The processed_run directory contains a number of plots and dataframes which should be self-explanatory. The main dataframe is processed_run/energies_df.csv and contains the computed energies for each variant in the run.

You can specify multiple run directories to process at a time by separating them with a space when calling process_run.py.

Add results to a database

You can add the results of one or more HTCondor runs to an SQLite database using the process_run.py script.

First, create the blank database using database.py.

python code/database.py create --db_fn variant_database/my_database.db

Then, add the results of one or more HTCondor runs to the database using the database mode of process_run.py.

python code/process_run.py database --main_run_dirs output/htcondor_runs/my_condor_run --db_fn variant_database/my_database.db

This database can now be used with the metl repository to create a processed Rosetta dataset and pretrain METL models.