E. H and J, cyan and gray; and S8), Genz-123346 Trp- 982.60 flips up and out of the binding pocket, pointing extracellularly, and the ligand moves between helices I and STMN1 II. This conformation of Trp- 982.60 more closely resembles CCR9 (and S8) and is the most prominent position of the residue as it extends deeper into the chemokine binding site toward helix III. In this case, the ligand interacts with helices II, IV, and V, and there are no transitions from this state to a dissociated state. As in the apo MSM, the absence of the orthosteric ligand causes a shift in the position Genz-123346 of Trp- 982.60. In the holo simulations shown in and for full description of materials and methods. MD trajectories and MSM construction scripts are available for download (33). System Preparation and Molecular Dynamics Simulations. Two systems were simulated for a total of 260 math xmlns:mml=”http://www.w3.org/1998/Math/MathML” id=”i8″ mi mathvariant=”normal” /mi /math s: CCR2 holo, with both cocrystallized antagonist ligands bound, and CCR2 apo, without ligands bound. CCR2-RA-[R] and BMS 681 (11) were removed to build the apo system. Each all-atom system is embedded in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer, explicitly solvated with transferrable intermolecular potential with 3 points (TIP3P) and simulated with 150 mM NaCl, at pH 7.4, at 310 K and 1 bar. The initial coordinates were taken from the experimental crystal structure (11). Building the MSMs. The MSMs were built with PyEMMA version 2.5.4 (58), selected based on implied timescale plots ( em SI Appendix /em , Fig. S14) and ChapmanCKolmogorov assessments ( em SI Appendix /em , Figs. S15 Genz-123346 and S16), and coarse grained with hidden Markov models (HMMs). Representative structures were selected from each macrostate by taking the centroid of the most populated microstate ( em SI Appendix /em , Figs. S17CS19). Supplementary Material Supplementary FileClick here to view.(48M, pdf) Acknowledgments We thank Tracy Handel and Irina Kufareva for their valuable input and initial modification of CCR2 coordinates. We thank Frank Noe and Cecilia Clementi for their useful discussions regarding MSM construction. We also thank the organizers and participants of the 2018 Workshop on Free Energy Methods, Kinetics and Markov State Models in Drug Design for helpful input. This work was supported in part by the Directors New Innovator Award Program NIH Grant DP2-OD007237, the National Biomedical Computation Resource NIH Grant P41-GM103426, and the National Science Foundation Genz-123346 through The Extreme Science and Engineering Discovery Environment (XSEDE) supercomputing resources provided via Award TG-CHE060073 (to R.E.A.). C.T.L. also acknowledges support from the NIH Molecular Biophysics Training Program (Grant T32-“type”:”entrez-nucleotide”,”attrs”:”text”:”GM008326″,”term_id”:”218382999″,”term_text”:”GM008326″GM008326). Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through Grant R01GM116961 from the NIH. The Anton 2 machine at PSC was generously made available by D. E. Shaw Research. Footnotes Conflict of interest statement: R.E.A. has equity interest in, and is a cofounder and on the scientific advisory board of Actavalon, Inc. This article is usually a PNAS Direct Submission. Data deposition: MD trajectories along with MSM construction and other analysis scripts have been deposited at the UC San Diego Library Digital Collections (https://doi.org/10.6075/J0QZ289Q). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1814131116/-/DCSupplemental..