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In this study, we present a proton-detected solid-state NMR (ssNMR) approach, combined with an optimized random partial protonation (RAP) labeling strategy, to determine the high-resolution structure of the large-conductance mechanosensitive channel (MscL) directly within native\n                  <jats:italic>E.coli<\/jats:italic>\n                  membranes (backbone RMSD = 1.9 \u00c5). Our approach effectively suppresses background protein signals and achieves high spectral resolution and sensitivity at moderate MAS frequencies (40\u201360 kHz) by differentially tuning amide and side-chain protonation levels. Using advanced recoupling schemes, we obtained chemical shift assignments of side-chain protons by 3D hCCH spectra and\n                  <jats:sup>1<\/jats:sup>\n                  H-\n                  <jats:sup>1<\/jats:sup>\n                  H distance restraints from a series of 3D hNHH spectra. With 10% protonation in side-chains, the\n                  <jats:sup>1<\/jats:sup>\n                  H signals displayed linewidths of approximately 50Hz, facilitating the extraction of 49 long-range distance restraints between amide and side-chain protons, which are crucial for structural convergence. Ambiguities in the assignment of weak signals corresponding to distance restraints were resolved by integrating 3D hNHH experimental data with CS-Rosetta structural modeling. The resulting structure reveals a well-defined pentameric assembly, with transmembrane helix packing consistent with that observed in detergent environments. This study demonstrates significant sensitivity advantages of\n                  <jats:sup>1<\/jats:sup>\n                  H-detected over\n                  <jats:sup>13<\/jats:sup>\n                  C-detected\n                  <jats:italic>in situ<\/jats:italic>\n                  ssNMR methods, highlighting the potential of\n                  <jats:sup>1<\/jats:sup>\n                  H-detected ssNMR for the structure determination of a broad range of membrane proteins in native membranes.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Abstract Figure<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"685061v2_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.10.28.685061","type":"posted-content","created":{"date-parts":[[2025,10,29]],"date-time":"2025-10-29T21:00:10Z","timestamp":1761771610000},"source":"Crossref","is-referenced-by-count":0,"title":["<i>In Situ<\/i>\n                  Structure Determination of a Membrane Protein in Native Cellular Membranes by Proton-Detected Solid-State NMR"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2865-6557","authenticated-orcid":false,"given":"Huayong","family":"Xie","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weijing","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hang","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yan","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yang","family":"Shen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuefang","family":"Gan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiong","family":"Tong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yongxiang","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huan","family":"Tan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun","family":"Yang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025103120550820000_2025.10.28.685061v2.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biophys-083012-130326"},{"key":"2025103120550820000_2025.10.28.685061v2.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2010.07.005"},{"key":"2025103120550820000_2025.10.28.685061v2.3","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.8b00538"},{"issue":"3","key":"2025103120550820000_2025.10.28.685061v2.4","doi-asserted-by":"crossref","first-page":"183533","DOI":"10.1016\/j.bbamem.2020.183533","article-title":"A 10-Year Meta-Analysis of Membrane Protein Structural Biology: Detergents, Membrane Mimetics, and Structure Determination Techniques","volume":"1863","year":"2021","journal-title":"Biochim. 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