The DBCO concentration indicates the final concentration of DBCO-sulfo-N-hydroxysuccinimide (NHS) ester crosslinker in DBCO modification on bead surface. The other conditions, such as DNA handle length, membrane protein concentration, and detergent/lipid concentration, indicate those in DBCO-azide conjugation step of the single-molecule system assembly. Used buffers are a phosphate-buffered saline (100 mM sodium phosphate, 150 mM NaCl, and pH 7.3) and a Tris-buffered saline (50 mM Tris, 150 mM NaCl, and pH 7.5). The bicelle (C or G) consists of DMPC (or DMPG) lipid and CHAPSO detergent at a 2.5:1 molar ratio. The % indicates w/v %. The symbol O indicates successful conjugation between DBCO-modified beads and target membrane proteins. In this case, specifically tethered beads are found, and the force-extension curves of the molecular constructs are obtained (confirmed by three replicates). The symbol X indicates unsuccessful conjugation or entire nonspecific binding of beads to the surface. In this case, no data are obtained. See Figure 3—source data 1 for the full list. Abbreviations: DMPC, Dimyristoylphosphatidylcholine; DMPG, Dimyristoylphosphatidylglycerol; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate.