Workshop 2026 – Bioinformatics Centre

Hands-on session for protein structure prediction by AlphaFold3 and ESMFold
Course Instructors:
Dr. Sanjeet Kumar Mahtha, MK Bhan Postdoctoral Fellow
Sureshkumar V, Research Scholar
Dr. Debasisa Mohanty, Director

AlphaFold3

URL: https://alphafoldserver.com/

Monomer	Example 1	Example 2
Dimer	Example 1	Example 2
Protein + Ligand	Protein Sequence	Ligand: FAD
Protein + RNA	Sequences
Protein + DNA	Sequences
Protein + DNA + Ion	Sequences	Ion: Sodium
Protein + PTMs	You can play with example 1

ESMFold

URL: https://esmatlas.com/resources?action=fold

Monomer	Example 1	Example 2
Dimer– API and Web server do not support Dimer. But you may use ESMFold ColabFold Portal	https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/ESMFold.ipynb	http://pdbi.nii.ac.in/AF_ESM/7awv_Dimer.fa [OR] Run on local server

ESM – API

curl -X POST --data "GAMADIGSMDVLEYFERLKNRELAFVLDDLQLSDMVTRRGFSVIPFDDFDLAREDHPPAFVLVTRLDYHGKLMQAWETAKGISSHLSLAKFDTSPKSVEYSLDQLLSMDFAETLKRRGDYYDSVASTNRMEVVTPGAVLTCDFGNEIEIANNDVEMQKGWLYSVAEFFETSVINLEADRSSYTLNGDLCFTGLIYLCNRPDLKERASATMDELMRMSTRGRNVVSFVDNQIVRMELGGVDMTATLRELIVGKEREGSSTEFAMGCVEYPLAQDWTINSVMNEGSHGIHVGVGMGKEIPHMDFIAKGAELRIAESSDA" https://api.esmatlas.com/foldSequence/v1/pdb/ > 6zsv_Out.pdb

How to predict multiple structures using the script?
Demo fasta file – Multi_batch.fa
Download Script – ESM_script.py
Command: python ESM_script.py

# If biopython is not installed, you install it with “pip install biopython”.

import requests
from Bio import SeqIO
import os,sys
# Define the ESMFold API endpoint
ESMFOLD_API_URL = "https://api.esmfold.org/v1/predict"
input = "Multi_batch.fa"
for i, record in enumerate(SeqIO.parse(input, "fasta"), start=1):
    sequence_id = record.id
    sequence = str(record.seq)
    print(f"Processing sequence {i}: ID = {sequence_id}")
    cmd = "curl -X POST --data ""+sequence+"" https://api.esmatlas.com/foldSequence/v1/pdb/ > "+sequence_id.replace("|","_")+".pdb"
    #print(cmd)
    os.system(cmd)

How to run ESMFold on the local machine?

Option 1:
wget http://pdbi.nii.ac.in/AF_ESM/Script_ESM.sh
wget http://pdbi.nii.ac.in/AF_ESM/ESM_dim_ex.fa
sbatch Script_ESM.sh


Option 2:
module load esmfold/1.0.3
wget http://pdbi.nii.ac.in/AF_ESM/ESM_dim_ex.fa

## Command for Dimer Structure prediction
esm-fold -i ESM_dim_ex.fa -o output_folder -m /opt/apps/esmfiles/

How to run AlphaFold3 with ligand in the local machine?

Option 1:
wget http://pdbi.nii.ac.in/AF_ESM/Script_AF3_Lig.sh
sbatch Script_AF3_Lig.sh

Option 2
module load AlphaFold/AF3
mkdir AF3_Lig
cd AF3_Lig
cp /apps/run_alphafold.py .
wget http://pdbi.nii.ac.in/AF_ESM/AF3_input_test_wMSA.json
python run_alphafold.py --json_path AF3_input_test_wMSA.json --model_dir /opt/apps/af3_model --db_dir /opt/apps/AF3_DB --output_dir Output_AF3 --run_data_pipeline False

Additional Links:
PAE Viewer – https://pae-viewer.uni-goettingen.de/
PyMOL Download Link – https://pymol.org/#download
PyMOL License – https://pymol.org/ep/
PyMOL Tutorial – https://pymol.sourceforge.net/newman/userman.pdf
Chimera – https://www.cgl.ucsf.edu/chimera/download.html

Practice Files – Download (Needed for tomorrow’s session)

Further Reading – Comparison of Structure Prediction Methods and Comparison of AlphaFold2, AlphaFold3 and ESM Fold: Click here

More Details:-

AlphaFold2	Article – https://doi.org/10.1038/s41586-021-03819-2 GitHub Source Code – https://github.com/google-deepmind/alphafold
AlphaFold3	Article – https://doi.org/10.1038/s41586-024-07487-w GitHub Source Code – https://github.com/google-deepmind/alphafold3
ESMFold	Article – https://doi.org/10.1126/science.ade2574 GitHub Source Code – https://github.com/facebookresearch/esm
RoseTTAFold	Article – https://doi.org/10.1126/science.abj8754 GitHub Source Code – https://github.com/RosettaCommons/RoseTTAFold

Download Programs for SSH/SCP connection (Only for Windows Users)
PuTTY – Download
WinSCP – Download