RevMD-Aqua

Documentation Index

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Why Use This Engine?

In the documentation below, we will use Revilico’s RevMD-Aqua engine to simulate a protein in explicit solvent under physiologically relevant conditions, without any ligand present. This type of simulation captures the natural conformational dynamics of the protein, revealing how the structure breathes, which binding site regions are flexible, and which protein conformations are thermodynamically accessible. The resulting trajectory snapshots serve as input for ensemble docking and as a reference for assessing target druggability under dynamic rather than static conditions.

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Background

Protein structures determined by crystallography or cryo-EM represent a single low-energy snapshot of a dynamic system. In solution, proteins undergo continuous conformational fluctuations ranging from local side chain rotations to domain-level rearrangements, and these motions are fundamentally important for function, allostery, and ligand binding. A binding site that appears druggable in a crystal structure may be transiently open or closed in solution, and a cryptic site invisible in the static structure may be revealed during molecular dynamics. RevMD-Aqua simulates the protein in a periodic water box using GROMACS, generating a trajectory that captures this conformational diversity over nanosecond timescales.

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Simulation Pipeline

System Construction The protein structure is placed in a periodic simulation box, typically a dodecahedron or cubic geometry, with a minimum distance of 1.0 nm from the protein surface to the box edge in any direction. The box is then filled with explicit TIP3P water molecules and neutralized by adding sodium and chloride ions to a physiological concentration of 0.15 M NaCl. This solvation environment mimics the ionic strength and dielectric properties of the cellular aqueous environment. Force Field Assignment Each atom in the system is assigned parameters from the AMBER99SB-ILDN force field, which defines the potential energy function governing all interatomic interactions. The total potential energy of the system is the sum of bonded and non-bonded terms: $$E_{\text{total}} = E_{\text{bonds}} + E_{\text{angles}} + E_{\text{dihedrals}} + E_{\text{VDW}} + E_{\text{elec}}$$ The bonded terms use harmonic potentials for bonds ($k_b(r - r_0)^2$) and angles ($k_\theta(\theta - \theta_0)^2$) and a periodic potential for dihedrals. Non-bonded interactions use a Lennard-Jones potential for van der Waals forces and a Coulomb potential for electrostatics, both computed with particle-mesh Ewald (PME) summation for long-range accuracy. Forces on each atom are computed as $F_i = -\nabla_{r_i} E$, and Newton’s equations of motion are integrated using the leapfrog algorithm with a 2 fs timestep. Energy Minimization Before dynamics begin, a steepest descent energy minimization step removes steric clashes and unrealistic geometries introduced during system construction. The system is minimized until the maximum force on any atom falls below 1000 kJ/mol/nm. Equilibration Equilibration occurs in two phases. In the NVT phase (constant volume and temperature), the system is heated to 300 K using a velocity-rescaling thermostat while restraining protein heavy atoms to their initial positions. This allows water molecules and ions to relax around the protein surface. In the NPT phase (constant pressure and temperature), the pressure barostat (Parrinello-Rahman) is activated and the box dimensions equilibrate to the correct density at 1 bar. Both phases run for 100 ps. Production MD The unrestrained production simulation runs for the user-specified duration (default 100 ns). Coordinates are written to the trajectory file at regular intervals (typically every 10 ps), producing a set of protein snapshots that capture the natural conformational ensemble. Velocities are initialized from a Maxwell-Boltzmann distribution at 300 K.

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Analysis Outputs

RMSD (Root Mean Square Deviation) RMSD measures how much the protein structure deviates from the initial reference frame over time. It is computed for all backbone atoms after least-squares superposition: $$\text{RMSD}(t) = \sqrt{\frac{1}{N}\sum_{i=1}^{N} |r_i(t) - r_i^{\text{ref}}|^2}$$ A stable RMSD plateau indicates that the simulation has converged to an equilibrium ensemble. Rising RMSD indicates ongoing structural rearrangement. RMSF (Root Mean Square Fluctuation) RMSF measures the time-averaged positional flexibility of each residue around its mean position. High RMSF regions correspond to flexible loops, termini, and dynamic binding site elements. Low RMSF regions correspond to the rigid hydrophobic core. Radius of Gyration The radius of gyration $R_g = \sqrt{\sum m_i |r_i - r_{\text{cm}}|^2 / \sum m_i}$ monitors overall protein compactness over the simulation. Systematic changes in $R_g$ can indicate unfolding or large-scale conformational transitions. Binding Site Dynamics For each identified binding pocket, volume and accessibility are tracked over the trajectory using the same Voronoi tessellation approach applied in RevPocket. This reveals whether pockets are constitutively open, transiently formed, or progressively closed, directly informing the value of ensemble docking against the extracted snapshots. Trajectory Snapshots for Ensemble Docking The production trajectory is sampled at regular intervals to extract protein conformations for use as receptor inputs in RevMD-Bind ensemble docking. Snapshots are structurally clustered and representative frames from each cluster are exported as PDB files.

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Running the Engine

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Inputs

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Outputs

• Trajectory file: Full atomic trajectory for visualization and analysis
• RMSD plot: Backbone deviation from the starting structure over time
• RMSF plot: Per-residue flexibility colored on the 3D structure
• Radius of gyration plot: Protein compactness over simulation time
• Binding site volume plot: Pocket opening and closing dynamics
• Ensemble snapshots: Clustered PDB frames for downstream ensemble docking input
• Energy components: Potential energy, temperature, pressure, and density over time