PDBFixer

What is PDB Fixer?

PDBFixer repairs PDB and mmCIF structure files in preparation for molecular dynamics simulations. Part of the OpenMM toolkit, it adds missing heavy atoms, hydrogens, and entire loops; converts modified residues to standard amino acids; selects single conformations from alternate locations; deletes unwanted chains; and builds explicit solvent boxes or lipid membranes around the system.

Experimental structures from X-ray crystallography and cryo-EM rarely arrive simulation-ready. They lack hydrogens, contain incomplete side chains from regions of poor electron density, include crystallization artifacts like buffers and duplicate chains, and frequently carry modified residues (selenomethionine, phosphoserine) that standard force fields cannot parameterize. GROMACS, AMBER, and OpenMM all reject the raw files. PDBFixer addresses these issues through eight correction passes:

• Missing hydrogens: Adds all hydrogens absent from X-ray structures, with pH-dependent protonation
• Missing heavy atoms: Completes side chain atoms truncated by poor electron density
• Missing terminal atoms: Caps chain ends with the appropriate terminal atoms
• Missing residues: Builds complete loops in disordered regions using SEQRES records
• Nonstandard residues: Converts modified amino acids to their standard equivalents
• Heterogens: Selectively removes or preserves ligands, ions, and water
• Alternate locations: Picks a single conformation when multiple are recorded
• Solvent/membrane: Wraps the structure in a water box or lipid bilayer for explicit solvent simulations

ProteinIQ accepts one structure per job and returns the fixed structure with a run summary and applied-settings JSON file.

How to use PDB Fixer online

Upload a PDB or mmCIF file (or enter an RCSB PDB ID) to repair missing atoms and hydrogens. Rebuilding missing residues is optional and off by default. PDBFixer runs on cloud hardware with no local Python or OpenMM installation. Each job repairs one structure and returns its fixed file with a run summary.

Step 1: Load the structure

Upload one structure file or enter one RCSB PDB ID (1HSG for HIV-1 protease is a common test case). PDB (.pdb, .ent) and mmCIF/PDBx (.cif, .mmcif, .pdbx) formats are accepted, subject to the upload and atom limits shown for your account.

Step 2: Configure core processing

Under "Core processing," enable Prepare for molecular dynamics to standardize modified residues and use 0.15 M ionic strength when adding solvent. Leave it off when you want to control those settings individually. Keep Heterogens set to Keep all to preserve ligands, ions, and water. To extract a single chain from a multi-chain complex, list the unwanted chain IDs in Remove chains (e.g., B, C).

Step 3: Select what to add

Under "Add options," Add missing heavy atoms and Add missing hydrogens are enabled by default. Both are required for MD simulations. Enable Add missing residues only when complete chains are needed; this step is slow for structures with large gaps.

Step 4: Add solvent or membrane (optional)

For explicit solvent MD, enable Add solvent box; the default adds counterions needed for neutralization without extra salt. Set ionic strength to 0.15 M for physiological salt. For membrane proteins, enable Add lipid membrane instead; solvent and membrane are mutually exclusive.

Step 5: Run and download

Click Submit job to start processing. Once complete, the repaired structure can be downloaded directly or opened in the integrated PDB Viewer. A summary CSV and provenance JSON file record the requested and resolved settings.

Inputs and settings

Input requirements

Core processing

• pH value: Sets protonation states for titratable groups when adding hydrogens. Default 7.0. The field only applies when "Add missing hydrogens" is enabled.
• Output format: Writes either identifier-preserving PDB or PDBx/mmCIF, matching the two save formats in PDBFixer's desktop interface. Default PDB.
• Prepare for molecular dynamics: Enables nonstandard residue replacement and sets ionic strength to 0.15 M when adding solvent. Default Off.
• Heterogens: Keep all preserves ligands, ions, and water from the original structure. Keep only water removes ligands and ions but preserves crystallographic waters. Remove all strips all heterogens and keeps the polymer chains.
• Remove chains: Comma-separated chain IDs to exclude (e.g., B, C, D).
• Apply mutations: Point mutations introduced during processing. Format CHAIN:ORIGINAL-POSITION-NEW (e.g., A:ALA-57-GLY, B:VAL-123-ILE).

Add options

These settings control which structural elements PDBFixer adds to the input.

• Add missing residues: Builds entire missing loops from SEQRES records. Off by default. Slow for structures with large gaps (10+ residue loops).
• Missing residue regions: Optional comma-separated CHAIN:START-END selectors (for example A:45-52). Leave empty to add every region PDBFixer detects; a submitted selector that is not present in the structure is rejected with the available regions.
• Add missing heavy atoms: Completes truncated side chains. On by default. Required for most MD force fields.
• Add missing hydrogens: Adds hydrogens at the specified pH. On by default. Required for explicit hydrogen MD; can be disabled for rigid-body docking or implicit hydrogen force fields.
• Replace nonstandard residues: Converts modified amino acids (selenomethionine, phosphoserine) to their standard equivalents. Off by default to preserve deposited chemistry; enable it when standard force-field preparation requires standard residues.
• Selected residue replacements: Optional comma-separated CHAIN:RESIDUE_ID:NEW selectors (for example A:57:MET). Leave empty to apply every replacement PDBFixer suggests. Exact chain IDs and insertion codes are preserved.

Solvent box options

Adding explicit water creates a simulation-ready system. The protein is surrounded by a rectangular or truncated box of water molecules with counterions for charge neutralization.

• Add solvent box: Enables water box construction. Significantly increases processing time and output file size.
• Cation/Anion: Ion types for charge neutralization. Na+/Cl- is standard for most simulations. K+ is appropriate where potassium is physiologically relevant.
• Ionic strength: Molar concentration of added salt beyond the counterions needed to neutralize the system. Default 0.0 M matches PDBFixer; 0.15 M is common for physiological salt.
• Box geometry: Shape of the periodic boundary box when automatic padding is used. Cubic is standard and compatible with all software. Dodecahedron and Octahedron reduce water count by approximately 30% while maintaining minimum distance from protein to box edge, producing faster simulations with equivalent accuracy.
• Box sizing: Automatic padding (distance from protein to box edges) or explicit X/Y/Z dimensions in nanometers.
• Water model: TIP3P is the standard choice for most force fields. TIP4P-Ew provides improved density and diffusion properties. SPC/E is popular for GROMACS workflows.

Membrane options

Membrane systems embed the protein in a lipid bilayer for studying GPCRs, ion channels, and transporters.

• Add lipid membrane: Constructs a membrane system. Cannot be combined with Add solvent box; the membrane system includes water and ions automatically.
• Lipid type: Composition of the bilayer. POPC (palmitoyl-oleoyl-phosphatidylcholine) is the standard choice for general membrane protein studies. POPE is preferred for bacterial membranes.
• Membrane center Z: Position of the bilayer center along the Z-axis in nanometers. The default places the center at Z = 0; orient and position the input protein to span that plane. PDBFixer does not determine its membrane orientation automatically.
• Minimum padding: Distance from the protein to the box edges in nanometers.

Advanced options

• Solvent force field: Molecular mechanics force field used when constructing an explicit solvent box. PDBFixer default uses PDBFixer's standard force-field selection and can build definitions for retained nonstandard residues. Fixed AMBER14, AMBER99SB, and CHARMM36 choices require every retained residue to have compatible parameters; unsupported residues are rejected before solvation. Hydrogen placement, missing-heavy-atom repair, and membrane construction do not use this setting.
• Random seed: Seed passed to PDBFixer's missing-atom and missing-residue placement step. 0 lets PDBFixer choose its normal random state. This setting does not control hydrogen or solvent placement.
• Use custom box vectors: Manual triclinic box vectors (A, B, C as comma-separated X,Y,Z components in nanometers) instead of standard box shapes. Useful for non-orthogonal simulation setups.
• Download templates: Comma-separated residue codes to fetch from the PDB Chemical Component Dictionary (e.g., ATP, GTP, HEM). Use for non-standard residues not included in the default template library.

How does PDB Fixer work?

PDBFixer applies a stepwise workflow that compares each residue against template records and uses OpenMM's force fields to place missing atoms in chemically reasonable positions. It packages the most common preparation steps behind a single streamlined path.

Template-based correction

For each residue, PDBFixer consults the PDB Chemical Component Dictionary to determine which atoms should exist. Missing atoms are placed at ideal bond lengths, angles, and dihedrals defined in the template library. The deposited coordinates of resolved atoms are preserved exactly; only missing pieces are reconstructed.

Energy minimization

After placement, OpenMM minimizes the added heavy atoms while holding existing atoms fixed. This repair does not equilibrate the structure; simulation setup and equilibration remain separate steps.

Protonation states

When "Add missing hydrogens" is enabled, the pH value setting determines protonation. PDBFixer's source hydrogen-placement path preserves valid ligands and covalent modifications without requiring the whole structure to match the explicit-solvent force field. At pH 7.0, histidine residues are added neutral, aspartate and glutamate deprotonated, lysine and arginine protonated. Disulfide bonds are detected automatically and the bonded cysteines are kept neutral. For atypical pH (lysosomal pH 4.5, endosomal pH 5.5), adjust the setting accordingly; PROPKA can be run first to identify residues with shifted pKa values that warrant special attention.

Loop modeling

Missing residues are placed from residue templates along an initial connecting path and then minimized. These are approximate coordinates, not a prediction of the experimentally missing loop conformation. Inspect rebuilt regions before using them in further calculations.

Understanding the results

Every batch includes a downloadable run.log with the validated preparation settings, submitted/completed/failed structure counts, and bounded atom, residue, and chain summaries for successful results. Individual failure details and internal process diagnostics are kept out of this curated log.

Each job produces one fixed PDB or PDBx/mmCIF file in the selected format. The summary CSV records the source name, success status, output filename, atom and residue counts, processing steps applied, and any error message. The pdbfixer_settings_provenance.json file records tool versions plus the requested and resolved settings.

The 3D viewer labels each repaired model by its source name and shows one structure at a time, since PDBFixer does not generate alternative poses. Ligand-specific grouping controls are hidden for the same reason.

Validating the output

Visual inspection before long simulations catches problems that propagate into MD trajectories:

• Check that added loops adopt reasonable conformations with no severe clashes
• Verify that important ligands were not accidentally removed
• For membrane systems, confirm the protein spans the bilayer correctly

If added loops look unrealistic, extended energy minimization or brief MD equilibration refines the structure. The PDB Viewer provides in-browser inspection, and MolProbity delivers detailed geometry validation.

PDB Fixer vs manual preparation

Routine structure preparation involves several steps: identify missing atoms, fetch templates, add hydrogens, build solvent, and neutralize charges. PDBFixer automates these steps with selectable settings.

PDBFixer is well suited to routine structure processing and simulation setup. For complex cases requiring manual intervention (unusual ligands, specific protonation states, asymmetric membrane positioning), it serves as a strong starting point that can be refined in dedicated tools.

When to use PDB Fixer vs alternatives

Several tools overlap with PDBFixer on specific preparation tasks.

• PDB2PQR prepares structures for Poisson-Boltzmann electrostatics calculations and outputs PQR files with partial charges and atomic radii. The two tools address different downstream needs: PDBFixer for MD simulations, PDB2PQR for continuum electrostatics.
• OpenMM's Modeller is a lower-level API used by this workflow. It provides more granular control (custom residue templates, hybrid solvent setups) but requires Python scripting. Use it when the standard PDBFixer settings do not capture a required preparation step.
• CHARMM-GUI offers extensive options for membrane proteins, multicomponent systems, and NMR restraints through a web interface. It is heavier to use but produces CHARMM-native outputs and handles complex membrane insertion workflows that PDBFixer does not.
• AmberTools tleap is the standard preparation tool for AMBER force fields. It integrates directly with AMBER-specific parameter sets; the trade-off is less automation than PDBFixer.
• PDBe Motif, WHAT IF, and PDBSWS are web servers offering structure validation and repair. They are useful for one-off analysis but are not pipeline-friendly.
• PDBsum summarizes prepared structure features and interactions after coordinate repair.

For protonation state prediction at a specific pH, PROPKA provides pKa values that can guide the pH setting passed to PDBFixer. For binding site analysis on the fixed structure, fpocket identifies druggable pockets; for protein-ligand docking on the cleaned structure, AutoDock Vina and DiffDock are the standard starting points.

Common workflows

MD simulation preparation

Enable Prepare for molecular dynamics, then add a solvent box with 1.0 nm padding when explicit solvent is needed. With the default atom-repair controls enabled, this standardizes modified residues, adds missing heavy atoms and hydrogens, and uses 0.15 M ionic strength. The output PDB includes box vectors and counterions, ready for equilibration in OpenMM, GROMACS, or AMBER.

Docking preparation

Fix the structure with default settings, then use AutoDock Vina or DiffDock for molecular docking. Remove all deletes water, ions, and ligands. Keep all heterogens when the deposited ligand must remain as a reference pose, then remove only the molecules your docking workflow does not need.

Predicted structure cleanup

Structures from ESMFold, Boltz-2, AlphaFold 2, or OpenFold 3 often need protonation or format adjustments before downstream analysis. PDBFixer adds hydrogens and standardizes the structure.

Structure repair in workflows

In a workflow, each incoming structure is submitted as its own PDBFixer job. This keeps the source, settings provenance, fixed structure, and summary unambiguous for every result.

Best practices

• Start with default settings. Add missing heavy atoms and Add missing hydrogens handle most preparation needs without aggressive modification.
• Disable Add missing residues unless complete chains are required. Long gap fills produce approximate conformations needing extensive equilibration. If the missing region is not relevant to the study, leave it missing.
• Keep crystallographic waters at binding sites. These waters often mediate protein-ligand contacts and should be preserved when running docking or binding affinity calculations.
• Match ionic strength to the biological system. Keep 0 M for neutralization-only preparation, use 0.15 M for physiological salt, and consider 0.5 to 1.0 M for halophiles or aggregation studies.
• Use dodecahedral or octahedral boxes to save compute. They hold roughly 30% fewer waters than cubic boxes at the same minimum padding distance.
• Match water model to force field. TIP3P works with AMBER14 and CHARMM36; SPC/E is common in GROMACS workflows. Mixing incompatible water models with force fields produces incorrect thermodynamic properties.
• Choose lipid type by membrane origin. POPC for general eukaryotic membranes, POPE for bacterial inner membranes.
• Validate fixed structures before long simulations. Visual inspection with PDB Viewer catches missing ligands, broken loops, and protonation errors that propagate into MD trajectories.

Frequently asked questions

Is PDB Fixer free?

PDBFixer is open-source software licensed under MIT/LGPL. ProteinIQ provides a web interface with a free account tier and paid tiers for larger structures.

How long does PDB Fixer take to process a structure?

Small proteins (under 5,000 atoms) with default settings often finish quickly. Larger structures, missing-residue rebuilding, and solvent or membrane construction take longer on CPU, and structures with extensive loop building or large solvent boxes can take several minutes or more.

Why are my missing loops in the wrong conformation?

PDBFixer builds loops with idealized geometry followed by brief energy minimization. Long loops (10+ residues) often need additional refinement, such as extended minimization or short MD equilibration, to relax into reasonable conformations.

What is the difference between PDB Fixer and PROPKA?

PDBFixer adds missing atoms and prepares structures for simulation. PROPKA predicts pKa values for titratable residues without modifying the structure. The two are complementary: running PROPKA first identifies residues with shifted pKa values, and the pH setting passed to PDBFixer is then adjusted accordingly.

Can PDB Fixer output be used directly for MD simulations?

The output supplies prepared coordinates and, when solvent or membrane is added, periodic box information. You still need a compatible simulation force field, parameters for retained ligands, a simulation topology, and an equilibration protocol.

Why was a ligand removed?

The Heterogens setting controls this. Remove all strips all non-protein molecules, including ligands. Keep all preserves them along with crystallographic waters.

How are nonstandard amino acids handled?

Replace nonstandard residues converts modified residues to their standard equivalents when enabled. It is off by default to preserve modifications, but standard force fields may not parameterize them. Download templates can fetch specific residue definitions from the PDB Chemical Component Dictionary for custom ligands.

What force field should be used?

Use PDBFixer default for the broadest source-compatible solvent preparation, especially when retaining ligands or modified residues. Choose AMBER14, AMBER99SB, or CHARMM36 only when the entire retained structure is parameterized by that force field. The selection applies only to explicit solvent setup; hydrogen placement, missing-heavy-atom repair, and membrane construction follow PDBFixer's own behavior.

Are multiple chains supported?

Yes. PDBFixer processes all chains in the input structure. Remove chains excludes specific chains from the output.

How are membrane proteins prepared?

Enable Add lipid membrane rather than Add solvent box. Select the appropriate lipid type (POPC for general eukaryotic membranes, POPE for bacterial inner membranes) and set the membrane center to position the transmembrane domain correctly.