# A Self-Consistent Approach to Rotamer and Protonation State Assignments (RAPA): Moving Beyond Single Protein Configurations

## TL;DR

This paper introduces RAPA, a new method to determine multiple possible rotamer and protonation states in protein structures, improving accuracy beyond single-state predictions.

## Contribution

RAPA identifies multiple energetically consistent rotamer and protonation states for proteins, moving beyond single-state assignments.

## Key findings

- RAPA finds multiple rotamer and protonation states consistent with X-ray structures for most proteins.
- Most proteins have 8 or fewer energetically accessible configurations, avoiding combinatorial explosion.
- Molecular dynamics simulations confirm the stability of RAPA-predicted configurations.

## Abstract

There are currently over 160,000 protein crystal structures
obtained
by X-ray diffraction with resolutions of 1.5 Å or greater in
the Protein Data Bank. At these resolutions hydrogen atoms do not
resolve and heavy atoms such as oxygen, carbon, and nitrogen are indistinguishable.
This leads to ambiguity in the rotamer and protonation states of multiple
amino acids, notably asparagine, glutamine, histidine, serine, tyrosine,
and threonine. When the rotamer and protonation states of these residues
change, so too does the electrochemical surface of a binding site.
A variety of computational approaches have been developed to assign
states for these residues by investigating all possibilities and typically
deciding on a single rotamer or protonation state for each residue
that is consistent with the crystal structure. Here, we posit that
there are multiple rotamer and protonation states that are consistent
with the resolved structure of the proteins and introduce a Rotamer
and Protonation Assignment (RAPA) protocol which analyzes local hydrogen-bonding
environments in the resolved structures of proteins and identifies
a set of unique rotamer and protonation states that are energetically
consistent with the experimentally reported crystal structure. We
evaluate the RAPA-predicted configurations in molecular dynamics simulations
and find that there are multiple configurations for each protein that
maintain structures consistent with the X-ray results. In our initial
evaluations of the RAPA protocol, we find that for most proteins (69/77)
there are multiple energetically accessible rotamer and protonation
state configurations however the total number is limited to 8 or fewer
for most of the proteins (62 of 77). This suggests that there is no
combinatorial explosion in the number of energetically accessible
rotamer and protonation states for most proteins and investigating
all such states is computationally feasible.