Appendix 1—key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-FLAG M1 (mouse polyclonal IgG2a) | Gift from Prof. Brian Kobilka (PMID::17962520) |
Antibody was used to make anti-FLAG mAb resin that was used for the purification of FLAG-tagged M4 mAChR | |
| Strain, strain background (Escherichia coli) | DH5α | New England Biolabs | C2987H | |
| Strain, strain background (E. coli) | DH10bac | Thermo Fisher Scientific | 10361012 | |
| Cell line (Spodoptera frugiperda) | Sf9 | Expression Systems | 94-001S | |
| Cell line (Trichoplusia ni) | Tni | Expression Systems | 94-002S | |
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR WT | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | CHO K1 mouse M4 mAChR WT | PMID:21198541 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR D432E | This study | pEF5-FRT-V5-DEST plasmid | |
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR T433R | This study | pEF5-FRT-V5-DEST plasmid | |
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR V91L, D432E, T433R |
This study | pEF5-FRT-V5-DEST plasmid | |
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Y89A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Q184A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR F186A | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR W435A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR W439A | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR S85A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Y89A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Y92A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR I93T, I94V, K95I | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR I93T | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR I94V | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR K95I | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Y97A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR W98A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR G101A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR D106A | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR W108A | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR L109A | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR D112E | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR D112N | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR S116A | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR N117A | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR V120A | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR D129E | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR D129N | PMID:21300722 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR W164A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR F170A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR W171A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Q172A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR F173A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Q184A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR F186A | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR I187A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Q188A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR F189A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR L190A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR W413A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Y416A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR N423A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Q427A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR S428P | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR D432N | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR W435A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Y439A | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Y440A | PMID:26958838 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR C442A | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO human M4 mAChR Y443A | PMID:20406819 | ||
| Cell line (Chinese hamster ovary) | Flp-In CHO cell line | Thermo Fisher Scientific | R75807 | |
| Cell line (Homo sapiens) | 293A cell line | Thermo Fisher Scientific | R70507 | |
| Recombinant DNA reagent | Human FLAG-M4∆i3-His | This study | pVL1392 vector | |
| Recombinant DNA reagent | Human Gαi1 dominant negative mutant | PMID:32193322 | pFastBac vector | |
| Recombinant DNA reagent | Human Gβ1γ2 | Gift from Prof. Brian Kobilka (PMID:24256733) |
pVL1392 vector | |
| Recombinant DNA reagent | scFv16 | PMID:30213947 | pFastBac vector | |
| Recombinant DNA reagent | pcDNA5/FRT/TO-GAlphai1-RLuc8 | Gift from Prof. Bryan Roth (PMID:32367019) |
TRUPATH assay | |
| Recombinant DNA reagent | pcDNA3.1-Beta3 | Gift from Prof. Bryan Roth (PMID:32367019) |
TRUPATH assay | |
| Recombinant DNA reagent | pcDNA3.1-GGamma9-GFP2 | Gift from Prof. Bryan Roth (PMID:32367019) |
TRUPATH assay | |
| Chemical compound, drug | Acetylcholine | Sigma-Aldrich | ||
| Chemical compound, drug | Iperoxo | Sigma-Aldrich | ||
| Chemical compound, drug | LY2033298 | Sigma-Aldrich | ||
| Chemical compound, drug | VU0467154 | Gift from Prof. Craig Lindsley (PMID:25137629) | PMID:25137629 | |
| Chemical compound, drug | Prolume Purple | Nanolight Technology | ||
| Chemical compound, drug | [3H]-NMS | PerkinElmer | ||
| Chemical compound, drug | Polyethylenimine (PEI) | Sigma-Aldrich | ||
| Chemical compound, drug | Atropine | Sigma-Aldrich | ||
| Commercial assay or kit | AlphaScreen SureFire pERK 1/2 (Thr202/Tyr204) Assay Kits |
PerkinElmer | ||
| Software, algorithm | Prism 8.0 | GraphPad | ||
| Software, algorithm | PyMOL | Schrödinger | ||
| Software, algorithm | GaMD | PMID:26300708 | ||
| Software, algorithm | AMBER20 | https://ambermd.org | ||
| Software, algorithm | CPPTRAJ | PMID:26300708 | ||
| Software, algorithm | PyReweighting | PMID:25061441 | ||
| Software, algorithm | Phenix suite | PMID:20124702 | ||
| Software, algorithm | Coot | PMID:31730249 | ||
| Software, algorithm | Chimera | PMID:15264254 | ||
| Software, algorithm | Chimera X | PMID:32881101 | ||
| Software, algorithm | cryoSPARC | PMID:28165473 | ||
| Software, algorithm | Relion 3.1 | PMID:30412051 | ||
| Software, algorithm | Motioncor2 | PMID:28250466 | ||
| Software, algorithm | GCTF | PMID:26592709 | ||
| Software, algorithm | crYOLO | PMID:31240256 | ||
| Software, algorithm | ISOLDE | PMID:29872003 | ||
| Software, algorithm | MolProbity | PMID:29067766 | ||
| Software, algorithm | DAQ score | PMID:35953671 |