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	2.5	Å	Hydrogen-acceptor distance cutoff
D-H…A Angle	120.0	degrees	Donor-hydrogen-acceptor angle cutoff
D…A Distance	3.5	Å	Donor-acceptor distance cutoff
Weak H…A Distance	3.6	Å	Weak hydrogen-acceptor distance cutoff (C-H···O)
Weak D-H…A Angle	150.0	degrees	Weak donor-hydrogen-acceptor angle cutoff
Weak D…A Distance	3.5	Å	Weak donor-acceptor distance cutoff
X…A Distance	3.9	Å	Halogen-acceptor distance cutoff
C-X…A Angle	150.0	degrees	Carbon-halogen-acceptor angle cutoff
H…π Distance (legacy)	3.5	Å	Hydrogen-π center distance cutoff (legacy parameter)
D-H…π Angle (legacy)	110.0	degrees	Donor-hydrogen-π angle cutoff (legacy parameter)
C-Cl…π Distance	3.5	Å	Chlorine-π center distance cutoff
C-Cl…π Angle	145.0	degrees	Carbon-chlorine-π angle cutoff
C-Br…π Distance	3.5	Å	Bromine-π center distance cutoff
C-Br…π Angle	155.0	degrees	Carbon-bromine-π angle cutoff
C-I…π Distance	3.6	Å	Iodine-π center distance cutoff
C-I…π Angle	165.0	degrees	Carbon-iodine-π angle cutoff
C-H…π Distance	3.5	Å	Carbon-hydrogen-π distance cutoff
C-H…π Angle	110.0	degrees	Carbon-hydrogen-π angle cutoff
N-H…π Distance	3.2	Å	Nitrogen-hydrogen-π distance cutoff
N-H…π Angle	115.0	degrees	Nitrogen-hydrogen-π angle cutoff
O-H…π Distance	3.0	Å	Oxygen-hydrogen-π distance cutoff
O-H…π Angle	115.0	degrees	Oxygen-hydrogen-π angle cutoff
S-H…π Distance	3.8	Å	Sulfur-hydrogen-π distance cutoff
S-H…π Angle	105.0	degrees	Sulfur-hydrogen-π angle cutoff
PDB Fixing Enabled	True	boolean	Enable automatic structure fixing
PDB Fixing Method	“pdbfixer”	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)
C-H···O interactions: 2.8 - 3.6 Å (weak but significant)

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

Weak Hydrogen Bond Parameters (C-H···O)#

HBAT includes specific parameters for weak hydrogen bonds, particularly C-H···O interactions, which are important in protein structures and protein-ligand interactions.

H…A Distance Cutoff (Default: 3.6 Å)#

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

Physical significance:

Longer than conventional H-bonds due to weaker C-H donor
Accommodates the weak electrostatic nature of C-H bonds
Critical for detecting aromatic C-H donors

Typical ranges:

Aromatic C-H donors: 2.8 - 3.4 Å
Aliphatic C-H donors: 3.0 - 3.6 Å
Constrained geometries: up to 3.6 Å

D-H…A Angle Cutoff (Default: 150°)#

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

Physical significance:

More permissive than strong H-bonds (150° vs 120°)
Reflects the less directional nature of C-H···O interactions
Allows detection of geometrically constrained interactions

Examples of C-H···O Interactions:

Aromatic C-H of Phe, Tyr, Trp with backbone carbonyls
Methyl C-H groups with polar acceptors
Important in drug-protein binding interfaces
Contribute to protein stability and ligand binding affinity

D…A Distance Cutoff (Default: 3.5 Å)#

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

Physical significance:

Acts as a geometric constraint for weak hydrogen bonds
Similar to regular H-bonds but accounts for C-H bond geometry
Prevents detection of unrealistic interactions

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: 3.9 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: 150°)#

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: 150° - 160°
Weak but detectable: 130° - 150°
HBAT default: 150° (balanced detection)

π Interaction Parameters #

HBAT now supports comprehensive π interaction analysis with specific parameters for different interaction subtypes. π interactions involve atoms interacting with aromatic ring systems (PHE, TYR, TRP, HIS).

Interaction Subtypes#

HBAT distinguishes between several types of π interactions:

Halogen-π interactions: C-Cl…π, C-Br…π, C-I…π
Hydrogen-π interactions: C-H…π, N-H…π, O-H…π, S-H…π

Halogen-π Interaction Parameters#

C-Cl…π Interactions (Default: 3.5 Å, 145°)

Distance: Cl…π centroid distance cutoff
Angle: C-Cl…π centroid angle cutoff
Chlorine forms moderate-strength π interactions
Common in halogenated drug compounds

C-Br…π Interactions (Default: 3.5 Å, 155°)

Bromine has larger electron cloud than chlorine
More directional interactions (higher angle cutoff)
Stronger halogen-π interactions than chlorine
Important in medicinal chemistry

C-I…π Interactions (Default: 3.6 Å, 165°)

Iodine forms the strongest halogen-π interactions
Highly directional (approaching linear geometry)
Longer distance cutoff due to larger van der Waals radius
Most polarizable halogen

Hydrogen-π Interaction Parameters#

C-H…π Interactions (Default: 3.5 Å, 110°)

Weak but ubiquitous interactions in protein structures
Important for protein-ligand binding and protein folding
Angle measured as C-H…π centroid angle
Critical for drug design applications

N-H…π Interactions (Default: 3.2 Å, 115°)

Stronger than C-H…π due to more polarized N-H bond
Common in backbone-aromatic interactions
Important in secondary structure stabilization
Found in protein-protein interfaces

O-H…π Interactions (Default: 3.0 Å, 115°)

Strongest hydrogen-π interactions
Often found in active sites and binding pockets
Can compete with conventional hydrogen bonding
Important in enzyme catalysis

S-H…π Interactions (Default: 3.8 Å, 105°)

Less common but significant in sulfur-containing residues
Longer distance due to larger sulfur radius
Important in Cys and Met interactions
Relevant for disulfide bond environments

Ring Centroid Calculation#

Average position of aromatic carbon atoms in the ring
Represents the center of π electron density
Used as interaction target for all π interactions
Calculated for PHE, TYR, TRP, and HIS residues

Geometric Relationships#

Halogen-π:     Carbon(C) — Halogen(X) ··· π Ring Centroid
                    ↳ C-X...π angle ↲
                        ↳ X...π distance ↲

Hydrogen-π:    Donor(D) — Hydrogen(H) ··· π Ring Centroid
                   ↳ D-H...π angle ↲
                       ↳ H...π distance ↲

Legacy Parameters (Maintained for Compatibility)#

H…π Distance Cutoff (Legacy: 3.5 Å) D-H…π Angle Cutoff (Legacy: 110°)

These parameters are maintained for backward compatibility but are superseded by the specific subtype parameters above. When using the GUI or CLI, the subtype-specific parameters take precedence.

Migration Note: Existing analysis scripts and presets will continue to work, but it’s recommended to update to the new subtype-specific parameters for more accurate interaction detection.

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: “pdbfixer”)#

Definition: Choose the method for structure fixing operations.

Available options:

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

See PDB Structure Fixing for more details on each method.

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

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:

0.55: 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 comprehensive preset management for optimizing analysis parameters for different scenarios.

Note

For detailed information about preset management, including GUI usage, CLI commands, and creating custom presets, see Presets Management.

Quick Reference:

GUI: Access presets via Settings → Manage Presets
CLI: Use --preset preset_name or --list-presets
Built-in presets: high_resolution, standard_resolution, low_resolution, nmr_structures, strong_interactions_only, drug_design_strict, membrane_proteins, weak_interactions_permissive

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

# Include weak C-H···O interactions
hbat protein.pdb --whb-distance 3.6 --whb-angle 150

# Focus on strong halogen bonds
hbat protein.pdb --xb-distance 3.5 --xb-angle 160

# Comprehensive π interaction analysis with subtypes
hbat protein.pdb --pi-ch-distance 3.8 --pi-nh-distance 3.5 --pi-ccl-distance 3.7

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#

Default Parameter Values#

H…A Distance Cutoff (Default: 3.5 Å)#

D-H…A Angle Cutoff (Default: 120°)#

D…A Distance Cutoff (Default: 4.0 Å)#

H…A Distance Cutoff (Default: 3.6 Å)#

D-H…A Angle Cutoff (Default: 150°)#

D…A Distance Cutoff (Default: 3.5 Å)#

X…A Distance Cutoff (Default: 3.9 Å)#

C-X…A Angle Cutoff (Default: 150°)#

Interaction Subtypes#

Halogen-π Interaction Parameters#

Hydrogen-π Interaction Parameters#

Ring Centroid Calculation#

Geometric Relationships#

Legacy Parameters (Maintained for Compatibility)#

Core PDB Fixing Parameters#

fix_pdb_enabled (Default: True)#

fix_pdb_method (Default: “pdbfixer”)#

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)#

Covalent Bond Detection Factor (Default: 0.85)#

Analysis Mode#

High-Resolution Structures (< 1.5 Å)#

Low-Resolution Structures (> 2.5 Å)#

NMR Structures#

Focusing on Strong Interactions Only#

Drug Design Applications#

Protein Stability Studies#

Membrane Protein Analysis#

Enzyme Mechanism Studies#

Using Preset Files#

Setting Custom Parameters#

Parameter Validation#

Hydrogen Bonds#

Halogen Bonds#

π Interactions#

Computational Methods#