Mutate Everything

By Jeffrey Ouyang-Zhang, Daniel J. Diaz, Adam Klivans, Philipp Krähenbühl

This repository is an official implementation of the paper Predicting a Protein’s Stability under a Million Mutations.

TL; DR. parallel decode thermodynamic stability of a protein under single and double mutations

Main Results

Method	Backbone	S669 Spearman	cDNA2 nDCG	Total Train time (GPU hours)	URL
Mutate Everything	ESM2	0.47	0.38	3	log1/2 log2/2 model model (single only)
Mutate Everything	AlphaFold	0.56	0.47	24	log1/2 log2/2 model model (single only)

Data

cDNA,ProTherm Multiple,s669 are available here. It can also be prepared:

pip install gdown
gdown 1akfHt1WLbC345DgZwZ5kLeMUADq9XB7y
unzip mutate_everything_data
mv mutate_everything_data data

Place in ./data. The data should be organized as follows:

data/
    s669/
        mutations/
            s669.csv
        fasta/
            1A0FA/
                1A0FA.fasta
            ...
        msa/
            1A0FA/
                1A0FA.a3m
            ...

Installation

We prepare the conda environment to run our code below:

git clone https://github.com/jozhang97/MutateEverything
cd MutateEverything

conda env create --name=mutate_everything -f environment.yml
source activate mutate_everything

git clone https://github.com/HazyResearch/flash-attention
cd flash-attention
git checkout 5b838a8bef
python3 setup.py install
cd ..

cd openfold
python setup.py install
cd ..

Installation tested with torch1.12.1+cuda11.3 on A100,A40,RTX6000. We fork openfold from checkout b5fa2ba with minor changes.

Usage

Evaluation

You can evaluate our pretrained Mutate Everything models. Replace 1gl5 with your target protein.

For ESM, install the model here and place at models/mutate_everything_esm.pth. Then run:

python test.py \
    --backbone esm2_t33_650M_UR50D \
    --resume models/mutate_everything_esm.pth \
    --output_dir example/1gl5_esm \
    --name 1gl5 \
    --seq example/1gl5.fasta

For AlphaFold, first prepare the MSA for your sequence. For example, you can use Colabfold to do so. Install the model here and place at models/mutate_everything_af.pth. Then run:

python test.py \
    --backbone af \
    --resume models/mutate_everything_af.pth \
    --output_dir example/1gl5_af \
    --name 1gl5 \
    --seq example/1gl5.fasta \
    --msa_dir example/1gl5_msa

Training

To train Mutate Everything with ESM backbone on 3 GPUs. Training has two steps: first train the single mutation model, then train the double mutation model.

torchrun --nproc_per_node=3 main_train.py \
    --batch_size 1 \
    --data_path data/cdna/mutations/cdna_train.csv \
    --eval_data_paths data/s669/mutations/s669.csv \
    --dist_eval \
    --backbone esm2_t33_650M_UR50D \
    --freeze_at 14 \
    --lambda_double 0. --lambda_single 1. \
    --eval_period 999 --epochs 20 --warmup_epochs 2 --save_period 20 \
    --disable_wandb \
    --output_dir logs/esm_single

torchrun --nproc_per_node=3 main_train.py \
    --batch_size 1 \
    --data_path data/cdna/mutations/cdna_train.csv \
    --eval_data_paths data/cdna/mutations/cdna2_test.csv,data/s669/mutations/s669.csv,data/protherm/mutations/protherm_multiple.csv \
    --dist_eval \
    --backbone esm2_t33_650M_UR50D \
    --freeze_at 14 \
    --eval_period 1000 --epochs 100 --warmup_epochs 10 --save_period 100 \
    --finetune logs/esm_single/checkpoint-19.pth \
    --disable_wandb \
    --output_dir logs/esm_double

Download pre-trained Alphafold from Openfold and place at models/finetuning_ptm_2.pt:

pip install awscli
aws s3 cp --no-sign-request --region us-east-1 s3://openfold/openfold_params/ ./models --recursive

To train Mutate Everything with AlphaFold backbone on 3 GPUs:

torchrun --nproc_per_node=3 main_train.py \
    --batch_size 1 \
    --finetune_backbone models/finetuning_ptm_2.pt \
    --data_path data/cdna/mutations/cdna_train.csv \
    --eval_data_paths data/s669/mutations/s669.csv \
    --dist_eval \
    --backbone af \
    --lambda_double 0. --lambda_single 1. \
    --eval_period 999 --epochs 20 --warmup_epochs 2 --save_period 20 \
    --disable_wandb \
    --output_dir logs/af_single

torchrun --nproc_per_node=3 main_train.py \
    --batch_size 1 \
    --finetune_backbone models/finetuning_ptm_2.pt \
    --data_path data/cdna/mutations/cdna_train.csv \
    --eval_data_paths data/cdna/mutations/cdna2_test.csv,data/s669/mutations/s669.csv,data/protherm/mutations/protherm_multiple.csv \
    --dist_eval \
    --backbone af \
    --eval_period 1000 --epochs 100 --warmup_epochs 10 --save_period 100 \
    --finetune logs/af_single/checkpoint-19.pth \
    --disable_wandb \
    --output_dir logs/af_double

License

This project builds heavily off of MAE and extensively uses OpenFold. Please refer to their original licenses for more details.

Disclaimer

We observe a cysteine stabilization bias when examining DMS predictions (cysteine is often predicted to be the most stabilizing substitution). We are unsure if this is an artifact from the training data but attempts to fix this bias lead to worse metrics on the test set. Use cysteine predictions with caution.

Citing Mutate Everything

If you find Mutate Everything useful in your research, please consider citing:

@article{ouyangzhang2023predicting,
  title={Predicting a Protein’s Stability under a Million Mutations},
  author={Ouyang-Zhang, Jeffrey and Diaz, Daniel J and Klivans, Adam and Kr{\"a}henb{\"u}hl, Philipp},
  journal={NeurIPS},
  year={2023}
}