Organophosphate (OP) conjugation of the human acetylcholinesterase (hAChE) active site leads to a deadly increase of the neurotransmitter acetylcholine in the nervous system. The slow reactivation rate of OP‐hAChE by current antidotes is a major problem in effective treatment. This is a possible consequence of sterically restricted antidote access to the conjugated phosphorus in already narrow active center gorge of hAChE emphasized by available static X‐ray structural snapshots. We have conducted inelastic neutron scattering (INS) experiments on both hAChE and paraoxon (POX) conjugated hAChE (POX‐hAChE) to better understand the influence of OP conjugation on hAChE dynamics and reactivation rate limitation. The measured dynamic structure factor S(q,ω) showed a statistically significant difference at high Q between the two samples. The corresponding vibrational density of states (DOS) indicated that the difference was a result of low frequency vibrations in the 30 to 50 cm−1 range. In order to identify the particular mode of motion that is affected by OP conjugation, we compared the three X‐ray structures of the POX‐conjugated hAChE with the apo‐hAChE, and found tertiary conformation deviations in three regions: the acyl pocket, C‐terminal helix bundle and the Ω loop. We then constructed computational models of the apo‐hAChE and POX‐hAChE powder samples each with eight randomly oriented monomers. After equilibration, multiple 10 ps simulations were performed to compute the vibrational DOS via the velocity autocorrelation functions of the protein heavy atoms. An additional set of 10 ps simulations were given an impulse to each of the three specific regions. These simulations confirmed that a motion closing the acyl loop gorge was in line with experimental INS data. This motion was further shown to be coupled with the helix bundle dynamics, providing an explanation for the change in hAChE quaternary structure after OP‐conjugation observed in the SAXS data. Taken together, our results reveal detailed molecular insights into how OP‐conjugation changes the dynamic behavior of hAChEs.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.