{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:48:21Z","timestamp":1792788501900,"version":"4.5.0"},"posted":{"date-parts":[[2026,9,8]]},"group-title":"Biophysics","reference-count":62,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["DA063209"],"award-info":[{"award-number":["DA063209"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"name":"St. Jude Children\u2019s Research Hospital Collaborative Research Consortium on GPCRs"},{"DOI":"10.13039\/100006108","name":"National Center for Advancing Translational Sciences","doi-asserted-by":"crossref","award":["UL1TR004419"],"award-info":[{"award-number":["UL1TR004419"]}],"id":[{"id":"10.13039\/100006108","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["1R24GM154042"],"award-info":[{"award-number":["1R24GM154042"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Office of Research Infrastructure of the National Institutes of Health","award":["S10OD026880"],"award-info":[{"award-number":["S10OD026880"]}]},{"name":"Office of Research Infrastructure of the National Institutes of Health","award":["S10OD030463"],"award-info":[{"award-number":["S10OD030463"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  How ligands with different efficacies regulate G protein-coupled receptor signaling remains incompletely understood. Here, we combine Markov state modeling of nearly one millisecond of aggregate all-atom molecular dynamics simulations with time-resolved cryogenic-electron microscopy (cryo-EM) to reconstruct the most probable transition pathways connecting recently resolved structures of guanosine triphosphate (GTP)-bound \u03bc-opioid receptor (MOR)\u2013G\n                  <jats:sub>i1<\/jats:sub>\n                  complexes with the full agonist lofentanil (LFT) or partial agonist mitragynine pseudoindoxyl (MP). The models predict four previously unresolved intermediate conformations. Guided by these predictions, reanalysis of the cryo-EM particle ensemble identifies conformational populations consistent with all four intermediates. Remarkably, the largest ligand-dependent structural differences emerge within these intermediates rather than in the previously resolved states. LFT favors a predominantly sequential and kinetically efficient transition pathway, whereas MP stabilizes a broader intermediate ensemble along this pathway and slows progression toward later states. Together, these complementary approaches provide an enriched thermodynamic and kinetic description of MOR-mediated G protein activation, revealing that opioid efficacy is encoded, at least in part, in the differential stabilization and kinetics of transition intermediates extending beyond GTP binding but well before complete G protein dissociation.\n                <\/jats:p>","DOI":"10.64898\/2026.09.02.748988","type":"posted-content","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T21:55:12Z","timestamp":1788904512000},"source":"Crossref","is-referenced-by-count":1,"title":["Transition Intermediates Encode Ligand Efficacy at the \u03bc-Opioid Receptor-Gi Protein Complex"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8038-9105","authenticated-orcid":false,"given":"Kirill","family":"Konovalov","sequence":"first","affiliation":[{"name":"Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1434-0316","authenticated-orcid":false,"given":"Sabina","family":"Novack","sequence":"additional","affiliation":[{"name":"Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2868-303X","authenticated-orcid":false,"given":"Davide","family":"Provasi","sequence":"additional","affiliation":[{"name":"Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3861-7973","authenticated-orcid":false,"given":"Vishal","family":"Maingi","sequence":"additional","affiliation":[{"name":"Department of Structural Biology, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0238-7846","authenticated-orcid":false,"given":"Georgios","family":"Skiniotis","sequence":"additional","affiliation":[{"name":"Department of Structural Biology, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA"},{"name":"Center of Excellence for Structural Cell Biology, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4382-8276","authenticated-orcid":false,"given":"Marta","family":"Filizola","sequence":"additional","affiliation":[{"name":"Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091112151628000_2026.09.02.748988v1.1","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.tips.2024.12.007","article-title":"Advances in the structural understanding of opioid allostery","volume":"46","year":"2025","journal-title":"Trends Pharmacol Sci"},{"key":"2026091112151628000_2026.09.02.748988v1.2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202200269"},{"key":"2026091112151628000_2026.09.02.748988v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2022.09.041"},{"key":"2026091112151628000_2026.09.02.748988v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2022.12.026"},{"key":"2026091112151628000_2026.09.02.748988v1.5","doi-asserted-by":"crossref","first-page":"1490","DOI":"10.1021\/acscentsci.4c00525","article-title":"Signaling Modulation Mediated by Ligand Water Interactions with the Sodium Site at muOR","volume":"10","year":"2024","journal-title":"ACS Cent Sci"},{"key":"2026091112151628000_2026.09.02.748988v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-025-01191-8"},{"key":"2026091112151628000_2026.09.02.748988v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-47792-6"},{"key":"2026091112151628000_2026.09.02.748988v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0219-7"},{"key":"2026091112151628000_2026.09.02.748988v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-05588-y"},{"key":"2026091112151628000_2026.09.02.748988v1.10","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1038\/s41586-025-09677-6","article-title":"Structural snapshots capture nucleotide release at the mu-opioid receptor","volume":"648","year":"2025","journal-title":"Nature"},{"key":"2026091112151628000_2026.09.02.748988v1.11","doi-asserted-by":"crossref","first-page":"794","DOI":"10.1038\/s41586-025-10056-4","article-title":"Non-Equilibrium Snapshots of Ligand Efficacy at the \u03bc-Opioid Receptor","volume":"652","year":"2026","journal-title":"Nature"},{"key":"2026091112151628000_2026.09.02.748988v1.12","unstructured":"Gomez, J.L. et al. 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