Analysis Parameters Guide#

This document provides comprehensive explanations of all analysis parameters used in HBAT for detecting and analyzing molecular interactions including hydrogen bonds, halogen bonds, and π interactions.

Overview #

HBAT uses geometric criteria to identify molecular interactions based on distance and angle cutoffs. These parameters are based on established literature values but can be customized based on your specific analysis needs.

Default Parameter Values #

Parameter	Default Value	Units	Description
H…A Distance	3.5	Å	Hydrogen-acceptor distance cutoff
D-H…A Angle	120.0	degrees	Donor-hydrogen-acceptor angle cutoff
D…A Distance	4.0	Å	Donor-acceptor distance cutoff
X…A Distance	4.0	Å	Halogen-acceptor distance cutoff
C-X…A Angle	120.0	degrees	Carbon-halogen-acceptor angle cutoff
H…π Distance	4.5	Å	Hydrogen-π center distance cutoff
D-H…π Angle	90.0	degrees	Donor-hydrogen-π angle cutoff
PDB Fixing Enabled	False	boolean	Enable automatic structure fixing
PDB Fixing Method	“openbabel”	string	Method for structure enhancement
Add Hydrogens	True	boolean	Add missing hydrogen atoms
Add Heavy Atoms	False	boolean	Add missing heavy atoms (PDBFixer only)
Replace Nonstandard	False	boolean	Convert non-standard residues (PDBFixer only)
Remove Heterogens	False	boolean	Remove non-protein molecules (PDBFixer only)
Keep Water	True	boolean	Preserve water when removing heterogens

Hydrogen Bond Parameters #

Hydrogen bonds are detected using three geometric criteria that must all be satisfied simultaneously.

H…A Distance Cutoff (Default: 3.5 Å)#

Definition: The direct distance between the hydrogen atom (H) and the acceptor atom (A).

Physical significance:

Represents the actual electrostatic interaction distance
Primary determinant of hydrogen bond strength
Based on van der Waals radii and experimental observations

Geometric relationship:

Donor(D) — Hydrogen(H) ··· Acceptor(A)
                ↳ H...A distance ↲

Typical ranges:

Strong H-bonds: 1.5 - 2.2 Å (e.g., O-H···O⁻)
Moderate H-bonds: 2.2 - 2.5 Å (e.g., N-H···O)
Weak H-bonds: 2.5 - 3.5 Å (e.g., C-H···O)

Examples:

Asp OD1···HN Val: H…A = 2.1 Å (strong)
Ser OG···HN Gly: H…A = 2.8 Å (moderate)
Tyr OH···O backbone: H…A = 3.2 Å (weak but significant)

D-H…A Angle Cutoff (Default: 120°)#

Definition: The angle formed by the donor atom (D), hydrogen atom (H), and acceptor atom (A).

Physical significance:

Ensures proper orbital overlap for hydrogen bonding
Reflects the directional nature of hydrogen bonds
More linear angles indicate stronger interactions

Geometric relationship:

       Acceptor(A)
          ↗
Donor(D) — Hydrogen(H)
     ↳ D-H...A angle ↲

Typical ranges:

Linear (strongest): 160° - 180°
Moderate: 140° - 160°
Weak but acceptable: 120° - 140°
Below 120°: Generally not considered hydrogen bonds

Examples:

Backbone N-H···O=C: ~165° (near linear, strong)
Side chain interactions: 130° - 150° (moderate)
Constrained geometries: 120° - 130° (weak)

D…A Distance Cutoff (Default: 4.0 Å)#

Definition: The distance between the donor heavy atom (D) and acceptor atom (A).

Physical significance:

Acts as a geometric constraint and pre-filter
Ensures reasonable overall hydrogen bond geometry
Prevents detection of unrealistically extended interactions

Geometric relationship:

Donor(D) — Hydrogen(H) ··· Acceptor(A)
    ↳ D...A distance ↲

Relationship to H…A distance:

D…A distance ≈ H…A distance + D-H bond length (~1.0 Å)
Should always be larger than H…A distance
Typical difference: 0.5 - 1.5 Å

Examples:

If H…A = 2.8 Å, then D…A ≈ 3.1 Å
If H…A = 3.2 Å, then D…A ≈ 3.5 Å

Halogen Bond Parameters #

Halogen bonds involve halogen atoms (F, Cl, Br, I) acting as electrophilic centers interacting with nucleophilic acceptors.

X…A Distance Cutoff (Default: 4.0 Å)#

Definition: The distance between the halogen atom (X) and the acceptor atom (A).

Physical significance:

Based on the sum of van der Waals radii
Halogen bonds are typically longer than hydrogen bonds
Larger halogens can form longer interactions

Halogen-specific typical ranges:

Fluorine: 2.6 - 3.2 Å
Chlorine: 3.0 - 3.6 Å
Bromine: 3.2 - 3.8 Å
Iodine: 3.4 - 4.0 Å

Examples:

Br···N His: 3.4 Å (strong halogen bond)
Cl···O backbone: 3.2 Å (moderate)
I···S Met: 3.8 Å (weak but significant)

C-X…A Angle Cutoff (Default: 120°)#

Definition: The angle formed by the carbon atom (C), halogen atom (X), and acceptor atom (A).

Physical significance:

Reflects the directionality of the σ-hole on the halogen
More linear angles indicate stronger halogen bonds
Based on the electron density distribution around halogens

Geometric relationship:

       Acceptor(A)
          ↗
Carbon(C) — Halogen(X)
      ↳ C-X...A angle ↲

Typical ranges:

Strong halogen bonds: 160° - 180°
Moderate: 140° - 160°
Weak but detectable: 120° - 140°

π Interaction Parameters #

π interactions involve hydrogen atoms interacting with aromatic ring systems (PHE, TYR, TRP, HIS).

H…π Distance Cutoff (Default: 4.5 Å)#

Definition: The distance between a hydrogen atom and the centroid of an aromatic ring.

Physical significance:

Represents the interaction between H and the π electron cloud
Generally longer than conventional hydrogen bonds
Includes both direct H…π and edge-to-face interactions

Ring centroid calculation:

Average position of aromatic carbon atoms
Represents the center of electron density

Typical ranges:

Strong π interactions: 2.4 - 3.2 Å
Moderate: 3.2 - 4.0 Å
Weak: 4.0 - 4.5 Å

Examples:

Arg NH···π Phe: 3.1 Å (cation-π interaction)
backbone NH···π Trp: 3.6 Å (moderate)
side chain OH···π Tyr: 4.2 Å (weak)

D-H…π Angle Cutoff (Default: 90°)#

Definition: The angle between the D-H bond vector and the vector from H to the π centroid.

Physical significance:

Different from other angle definitions (measures approach angle)
Smaller angles indicate more perpendicular approach to ring
Reflects optimal orbital overlap with π system

Geometric relationship:

    π Ring Centroid
         ↑
         |
Donor(D) — Hydrogen(H)
     ↳ D-H...π angle ↲

Angle interpretation:

0° - 30°: Perpendicular approach (optimal)
30° - 60°: Good π interaction geometry
60° - 90°: Acceptable but weaker
> 90°: Generally not considered π interactions

PDB Structure Fixing Parameters #

HBAT includes comprehensive PDB structure fixing capabilities to enhance analysis quality by adding missing atoms, standardizing residues, and cleaning structures. These parameters control automated structure preparation.

Note

For detailed information about PDB fixing methods and workflows, see PDB Structure Fixing.

Core PDB Fixing Parameters #

fix_pdb_enabled (Default: True)#

Definition: Enable or disable automatic PDB structure fixing.

Purpose:

Controls whether structure enhancement is applied before analysis
Must be enabled to access other PDB fixing features
Provides option to analyze original structures unchanged

Usage considerations:

Enable for: Crystal structures missing hydrogens, incomplete side chains
Disable for: Pre-processed structures, performance-critical workflows
Default disabled: Preserves original analysis behavior

fix_pdb_method (Default: “openbabel”)#

Definition: Choose the method for structure fixing operations.

Available options:

“openbabel”: Fast hydrogen addition, good for routine analysis
“pdbfixer”: Comprehensive fixing with advanced capabilities

Method comparison:

Capability	OpenBabel	PDBFixer
Add hydrogens	✓ Fast and reliable	✓ pH-dependent protonation
Add heavy atoms	✗ Not supported	✓ Complete missing atoms
Convert residues	✗ Limited	✓ Comprehensive database
Remove heterogens	✗ Not supported	✓ Selective removal
Speed	Very fast	Moderate
Dependencies	Lightweight	Requires OpenMM

fix_pdb_add_hydrogens (Default: True)#

Definition: Add missing hydrogen atoms to the structure.

Physical significance:

Most PDB crystal structures lack hydrogen atoms
Essential for accurate hydrogen bond analysis
Improves interaction detection completeness

Method-specific behavior:

OpenBabel: Standard hydrogen placement with chemical rules
PDBFixer: pH-dependent protonation states (His, Cys, Asp, Glu, Lys, Arg)

Impact on analysis:

Dramatically increases hydrogen bond detection
Enables complete interaction network analysis
Critical for meaningful cooperativity assessment

fix_pdb_add_heavy_atoms (Default: False, PDBFixer only)#

Definition: Add missing heavy atoms to complete incomplete residues.

Use cases:

Low-resolution structures with missing side chain atoms
Truncated residues in crystal contacts
Structures with disordered regions

Processing approach:

Identifies missing atoms using standard residue templates
Adds atoms with reasonable geometric placement
Preserves existing atom positions

Considerations:

May add atoms in energetically unfavorable positions
Requires subsequent energy minimization for accuracy
Useful for completeness rather than precision

fix_pdb_replace_nonstandard (Default: False, PDBFixer only)#

Definition: Convert non-standard amino acid residues to standard equivalents.

Common conversions:

MSE (selenomethionine) → MET (methionine)
CSO (cysteine sulfenic acid) → CYS (cysteine)
HYP (hydroxyproline) → PRO (proline)
PCA (pyroglutamic acid) → GLU (glutamic acid)

Benefits:

Ensures consistent analysis parameters
Prevents unrecognized residue errors
Enables standard interaction pattern recognition

Limitations:

May lose important chemical information
Could affect binding site analysis
Not suitable for studies focusing on modified residues

fix_pdb_remove_heterogens (Default: False, PDBFixer only)#

Definition: Remove non-protein heterogens (ligands, ions, etc.) from structure.

Removed by default:

Small molecule ligands
Metal ions
Crystallization additives
Buffer components

Interaction with keep_water:

When fix_pdb_keep_water is True: water molecules are preserved
When fix_pdb_keep_water is False: all heterogens including water are removed

Use cases:

Remove for: Clean protein-only analysis, secondary structure focus
Keep for: Binding site analysis, metal coordination studies

fix_pdb_keep_water (Default: True, PDBFixer only)#

Definition: When removing heterogens, preserve water molecules.

Rationale for keeping water:

Water mediates many protein interactions
Important for realistic hydrogen bond networks
Critical for binding site analysis

Rationale for removing water:

Simplifies analysis for protein-only studies
Reduces computational complexity
Focuses on direct protein interactions

Effect on analysis:

With water: More comprehensive interaction networks, water-mediated bonds
Without water: Direct protein interactions only, simplified patterns

Advanced PDB Fixing Parameters #

pH Parameter (PDBFixer method)#

Definition: pH value for determining protonation states (default: 7.0).

Affected residues:

Histidine: Protonation of ND1/NE2 based on pH
Aspartic acid: Protonation state of carboxyl group
Glutamic acid: Protonation state of carboxyl group
Lysine: Amino group protonation
Arginine: Guanidinium group state
Cysteine: Potential disulfide bond formation

pH ranges and effects:

Low pH (< 4): More protonated states, positively charged
Physiological pH (7.0): Standard protonation patterns
High pH (> 10): More deprotonated states, negatively charged

Setting guidelines:

pH 7.0: Standard for most protein analyses
pH 6.0: Slightly acidic conditions (some enzymes)
pH 8.0: Slightly basic conditions (alkaline phosphatases)

General Analysis Parameters #

Covalent Bond Detection Factor (Default: 0.85)#

Definition: Multiplier applied to Van der Waals radii sum for covalent bond detection.

Purpose:

Distinguishes between covalent bonds and non-covalent interactions
Accounts for the difference between Van der Waals and covalent radii
Prevents false positive interactions between bonded atoms

Calculation:

Bond cutoff = (VdW radius₁ + VdW radius₂) × factor

Valid range: 0.0 - 1.0

Typical values:

0.70: Very strict covalent bond detection
0.85 (default): Standard bond detection based on typical covalent/VdW ratio
1.00: Maximum permissive (uses full Van der Waals radii sum)

Analysis Mode #

Complete mode (default):

Analyzes all possible donor-acceptor pairs
Includes inter-residue and intra-residue interactions
Comprehensive analysis suitable for most applications

Local mode:

Only analyzes intra-residue interactions
Faster computation for large structures
Useful for studying local structural effects

Parameter Tuning Guidelines #

High-Resolution Structures (< 1.5 Å)#

Recommended adjustments:

H…A distance: 3.2 Å (stricter)
D-H…A angle: 130° (more stringent)
D…A distance: 3.7 Å (tighter constraint)

Rationale: High-resolution data allows for more precise geometric criteria.

Low-Resolution Structures (> 2.5 Å)#

Recommended adjustments:

H…A distance: 3.8 Å (more permissive)
D-H…A angle: 110° (more tolerant)
D…A distance: 4.3 Å (looser constraint)

Rationale: Coordinate uncertainty requires more tolerant criteria.

NMR Structures #

Recommended adjustments:

All distance cutoffs: +0.2 Å
All angle cutoffs: -10°
Consider ensemble averaging

Rationale: NMR structures have inherent flexibility and coordinate uncertainty.

Focusing on Strong Interactions Only #

Recommended adjustments:

H…A distance: 2.8 Å
D-H…A angle: 140°
X…A distance: 3.5 Å

Rationale: Identifies only the most significant interactions.

Common Use Cases #

Drug Design Applications #

Parameters:

Standard defaults with H…A ≤ 3.2 Å
Include halogen bonds (important for drug interactions)
Consider π interactions for aromatic compounds

Focus: Protein-ligand interfaces, binding site analysis

Protein Stability Studies #

Parameters:

Complete mode with standard defaults
Include all interaction types
Consider cooperativity chains

Focus: Secondary structure stabilization, fold stability

Membrane Protein Analysis #

Parameters:

Slightly more permissive due to lower resolution
H…A distance: 3.7 Å
Include π interactions (common in membrane environments)

Focus: Transmembrane regions, lipid-protein interactions

Enzyme Mechanism Studies #

Parameters:

Strict criteria for active site (H…A ≤ 3.0 Å)
Standard criteria for overall structure
Focus on cooperativity chains

Focus: Catalytic residues, substrate binding

Parameter Presets #

HBAT provides example parameter presets for common analysis scenarios, as well as the ability to save and load custom presets.

Example Presets #

The example_presets/ folder contains predefined parameter sets optimized for different structure types and analysis goals:

Preset File	Description	Use Case
🔬 high_resolution.hbat	Strict criteria for high-quality structures	X-ray structures with excellent resolution (< 1.5Å)
⚙️ standard_resolution.hbat	Default HBAT parameters	Most protein crystal structures (1.5-2.5Å)
📐 low_resolution.hbat	More permissive criteria	Lower resolution structures (> 2.5Å)
🧬 nmr_structures.hbat	Accounts for structural flexibility	Solution NMR structures
💪 strong_interactions_only.hbat	Very strict criteria	Focus on the strongest interactions
💊 drug_design_strict.hbat	Optimized for protein-ligand analysis	Drug discovery applications
🧱 membrane_proteins.hbat	Adapted for membrane environments	Transmembrane proteins
🌐 weak_interactions_permissive.hbat	Captures weak but significant interactions	Comprehensive interaction analysis

Preset Management #

Loading Example Presets#

Click “Load Preset…” button in the GUI
Navigate to the example_presets/ folder (opens by default)
Select the appropriate .hbat preset file
Parameters are automatically applied

Saving Custom Presets#

Configure your desired parameters in the GUI
Click “Save Preset…” button
Choose filename and location
The preset is saved as a .hbat file

Using Presets#

# Example: Load a preset and analyze
# 1. Open HBAT GUI
# 2. Load preset: example_presets/drug_design_strict.hbat
# 3. Load PDB file and run analysis

Preset File Format#

HBAT presets are saved as JSON files with the following structure:

{
  "format_version": "1.0",
  "application": "HBAT",
  "created": "2024-01-15T10:30:00.000000",
  "description": "Custom preset description",
  "parameters": {
    "hydrogen_bonds": {
      "h_a_distance_cutoff": 3.5,
      "dha_angle_cutoff": 120.0,
      "d_a_distance_cutoff": 4.0
    },
    "halogen_bonds": {
      "x_a_distance_cutoff": 4.0,
      "cxa_angle_cutoff": 120.0
    },
    "pi_interactions": {
      "h_pi_distance_cutoff": 4.5,
      "dh_pi_angle_cutoff": 90.0
    },
    "general": {
      "covalent_cutoff_factor": 0.85,
      "analysis_mode": "complete"
    },
    "pdb_fixing": {
      "enabled": false,
      "method": "openbabel",
      "add_hydrogens": true,
      "add_heavy_atoms": false,
      "replace_nonstandard": false,
      "remove_heterogens": false,
      "keep_water": true
    }
  }
}

Preset Storage Locations#

Example Presets (built-in):

Located in example_presets/ folder within the HBAT installation
Read-only preset files optimized for common scenarios

Custom Presets (user-created):

Windows: %USERPROFILE%\.hbat\presets\
macOS/Linux: ~/.hbat/presets/
Created when you save custom parameter configurations

Command Line Usage #

Using Preset Files #

# List all available presets
hbat --list-presets

# Use a specific preset
hbat protein.pdb --preset high_resolution
hbat protein.pdb --preset drug_design_strict
hbat protein.pdb --preset membrane_proteins

# Use preset with custom overrides
hbat protein.pdb --preset standard_resolution --hb-distance 3.2
hbat protein.pdb --preset nmr_structures --hb-angle 110 --da-distance 4.3

# Use custom preset file (full path)
hbat protein.pdb --preset /path/to/my_custom.hbat

# Use preset from current directory
hbat protein.pdb --preset my_custom.hbat

Preset Resolution Order:

If the preset name is an absolute path and exists, use it directly
If the preset name is a relative path and exists, use it from current directory
Look for the preset in the example_presets/ directory (with or without .hbat extension)
If not found, display an error and list available presets

Parameter Override Behavior:

When using --preset, the preset parameters are loaded first
Any additional CLI parameters will override the corresponding preset values
Only explicitly provided CLI parameters override preset values (not defaults)

Setting Custom Parameters #

# Strict hydrogen bond detection
hbat protein.pdb --hb-distance 3.2 --hb-angle 130 --da-distance 3.7

# Include weak interactions
hbat protein.pdb --hb-distance 3.8 --hb-angle 110 --da-distance 4.3

# Focus on strong halogen bonds
hbat protein.pdb --xb-distance 3.5 --xb-angle 140

# Comprehensive π interaction analysis
hbat protein.pdb --pi-distance 5.0 --pi-angle 100

Parameter Validation #

HBAT automatically validates parameter ranges:

Distance parameters: 0.1 - 10.0 Å
Angle parameters: 0.0 - 180.0°
Covalent factor: 0.5 - 3.0

Literature References #

Hydrogen Bonds #

Jeffrey, G.A. “An Introduction to Hydrogen Bonding” (1997)
Steiner, T. “The Hydrogen Bond in the Solid State” Angew. Chem. Int. Ed. 41, 48-76 (2002)
Donohue, J. “Selected Topics in Hydrogen Bonding” (1968)

Halogen Bonds #

Metrangolo, P. et al. “Halogen Bonding: Fundamentals and Applications” (2008)
Cavallo, G. et al. “The Halogen Bond” Chem. Rev. 116, 2478-2601 (2016)

π Interactions #

Meyer, E.A. et al. “Interactions with Aromatic Rings in Chemical and Biological Recognition” Angew. Chem. Int. Ed. 42, 1210-1250 (2003)
Salonen, L.M. et al. “Aromatic Rings in Chemical and Biological Recognition” Angew. Chem. Int. Ed. 50, 4808-4842 (2011)

Computational Methods #

McDonald, I.K. & Thornton, J.M. “Satisfying Hydrogen Bonding Potential in Proteins” J. Mol. Biol. 238, 777-793 (1994)
Hubbard, R.E. & Haider, M.K. “Hydrogen Bonds in Proteins” (2001)

For questions about parameter selection or custom analysis requirements, please refer to the HBAT documentation or open an issue on the GitHub repository.

Analysis Parameters Guide

Contents

Analysis Parameters Guide#

fix_pdb_enabled (Default: True)#

fix_pdb_method (Default: “openbabel”)#

fix_pdb_add_hydrogens (Default: True)#

fix_pdb_add_heavy_atoms (Default: False, PDBFixer only)#

fix_pdb_replace_nonstandard (Default: False, PDBFixer only)#

fix_pdb_remove_heterogens (Default: False, PDBFixer only)#

fix_pdb_keep_water (Default: True, PDBFixer only)#

pH Parameter (PDBFixer method)#

Loading Example Presets#

Saving Custom Presets#

Using Presets#

Preset File Format#

Preset Storage Locations#