How to Use an AlphaFold Structure for Molecular Dynamics Simulation

Load the AlphaFold PDB in PyMOL and immediately color by pLDDT (stored in the B-factor column). This gives you a spatial map of where to be careful before any other preparation step.

# Load and color by confidence
load AF-P04637-F1-model_v4.pdb
spectrum b, blue_cyan_yellow_orange_red, minimum=50, maximum=100
show cartoon

Use this confidence map to make decisions about every subsequent preparation step. Here’s how pLDDT translates to MD-specific decisions:

Remove signal peptides and propeptides

AlphaFold models the full UniProt sequence, which includes signal peptides, propeptides, and transit sequences that are cleaved in the mature protein. These should not be in your MD system — they’re not present in the biologically relevant form. Check the UniProt entry for your protein under PTM/Processing → Chain to identify the mature protein boundaries.

# Example: remove signal peptide residues 1-24
remove resi 1-24
save protein_mature.pdb

Decision: keep or remove low-pLDDT terminal regions

N- and C-terminal disordered tails (pLDDT below 50) should generally be removed before MD simulation if they are not the focus of your study. They add computational cost, increase the likelihood of simulation instability, and their dynamics are not meaningful because their starting positions are arbitrary.

To identify and remove them systematically in PyMOL:

# Find the first and last residues with pLDDT above 50
iterate (name CA and b > 50), print(resi)

# Remove terminal low-confidence residues (adjust range to your protein)
remove resi 1-12 or resi 488-500
save protein_cleaned.pdb

When to keep low-pLDDT regions

If your research question is specifically about a disordered region — a flexible linker, an intrinsically disordered regulatory domain, or a loop that you believe is functionally important — keep it in the simulation. But document clearly in your methods that the starting conformation of that region has pLDDT below 50 and should be considered arbitrary rather than a biologically meaningful starting point. The MD simulation itself will sample conformations for that region, which is more informative than the AlphaFold model.

Run pdb2gmx as you would for any protein, with two additional considerations specific to AlphaFold structures:

gmx pdb2gmx \
  -f protein_cleaned.pdb \
  -o protein.gro \
  -p topol.top \
  -water tip3p \
  -ff amber99sb-ildn \
  -ignh \
  -his

The -his flag is especially important for AlphaFold structures. Crystal structures often have pH-derived protonation state information from the crystallographic methods; AlphaFold models do not. GROMACS must assign protonation states to every histidine, and the automatic assignment may be wrong for active-site residues. The -his flag prompts you to assign each histidine manually — check your literature for known protonation states at pH 7.4, or run PropKa/H++ on the cleaned PDB first to get predictions.

AlphaFold structures may have more geometric imperfections than crystal structures — particularly in predicted loop regions and around the junction between well-folded and disordered segments. Use a more aggressive minimization protocol with a tighter convergence criterion:

integrator  = steep
emtol       = 500       ; stricter than standard 1000 kJ/mol/nm
emstep      = 0.01
nsteps      = 100000    ; more steps than standard 50,000

cutoff-scheme = Verlet
coulombtype   = PME
rcoulomb      = 1.2
rvdw          = 1.2
pbc           = xyz

Run minimization after solvation and ion addition (following the standard GROMACS preparation workflow):

gmx grompp -f minim.mdp -c system.gro -p topol.top -o em.tpr
gmx mdrun -v -deffnm em

If minimization does not converge even at 100,000 steps, a low-pLDDT region almost certainly has a severe geometric clash. Identify the problematic residue from the Fmax atom number reported in the log, then consider trimming or restraining that region more aggressively before re-running.

Standard MD equilibration uses position restraints on protein heavy atoms during NVT and NPT phases, then releases them for production. For AlphaFold structures, a graduated restraint release protocol is strongly recommended — especially for proteins with significant low-confidence regions.

The rationale: releasing all restraints simultaneously can cause low-pLDDT regions to undergo violent, unphysical motions in the early production phase, potentially destabilizing the well-folded core. Graduated release gives each part of the structure time to equilibrate in the presence of the force field before full freedom is granted.

A practical four-stage protocol:

• NVT equilibration — strong restraints on all backbone and side chain heavy atoms (1000 kJ/mol/nm² force constant). 200 ps.
• NPT equilibration phase 1 — strong backbone restraints (1000), weaker side chain restraints (500). 200 ps.
• NPT equilibration phase 2 — moderate backbone restraints (200), no side chain restraints. 200 ps. This stage is particularly important — it allows disordered regions to relax their side chains while backbone geometry is still guided.
• NPT equilibration phase 3 — weak backbone restraints (50) only. 200 ps. By this point most geometric tensions should be resolved.
• Production MD — no restraints. Standard run.

The production MD MDP file for an AlphaFold structure is identical to a crystal structure run — same force field, same timestep, same ensemble settings (Nosé-Hoover thermostat, Parrinello-Rahman barostat). Aim for at least 100 ns for a standard analysis; 200–500 ns gives more reliable flexibility profiles and convergence.

During analysis, track high-pLDDT and low-pLDDT regions separately:

# Create index group for high-confidence residues
# First identify which residues have pLDDT > 70 from the JSON
gmx make_ndx -f md.tpr -o highconf.ndx
# In the prompt: select residues known to be high-confidence
# Then calculate RMSD of only those residues
gmx rms -s md.tpr -f md_centered.xtc -n highconf.ndx -o rmsd_core.xvg -tu ns

Separating core RMSD from loop RMSD gives you a cleaner equilibration picture — the well-folded core should plateau within a few nanoseconds, while disordered loops may fluctuate throughout, which is expected and correct.