Abstract: Nucleotide binding domain, leucine-rich repeat, and pyrin domain-containing 3(NLRP3) is an NLR-protein family member that can be activated by diverse exogenous and endogenous stimuli but without direct binding of any of these pathogen ligands. Biological studies show that inactive NLRP3 is usually in an assembly state and its activation requires a kinase protein, NEK7. However, our recent computational studies as well as other biological investigations have demonstrated that NEK7 does not play a significant role in the activation of NLRP3 assembly and activation. Instead, biological studies suggest that NEK7 is essential in the dissociation of inactive NLRP3 assemblies. Despite extensive research, the dissociation mechanism of the inactive NLRP3 assembly remains largely elusive. In this work, an improved MM-PBSA method is applied to the protein-protein binding free energies in the inactive NLRP3 decamer. Combined with the potential mean force (PMF) computation for the 0\text°\to 5\text° conformational change, the standard free-energy change, \Delta G^0 is calculated for NEK7-driven association of the inactive NLRP3 decamer. Our calculations show that in the absence of NEK7, the dissociation of the inactive NLRP3 decamer is an energetically unfavorable process ( \Delta G^0=99.69\;\mathrmk\mathrmc\mathrma\mathrml/\mathrmm\mathrmo\mathrml ), whereas upon NEK7 binding, the overall standard free energy difference \Delta G^0=-24.21\;\rmkcal/mol is obtained for the inactive NLRP3 decamer dissociation. The free-energy difference calculations in this work also disclose an energetically optimized dissociation pathway, along which the inactive NLRP3 decamer is disunited by a one-by-one dissociation mechanism.