Materials and reagents

Biological materials

1. Active-state ADGRL4 (version CTF2B) HEK293 GnTI^- TetR stable cell line (stable suspension cell line with tetracycline inducible expression of the CTF2B active-state variant of ADGRL4. Available on request from Dr David Favara). Plasmids used to generate this stable inducible cell line (along with the detailed workflow for cell line development) are available on request from Dr David Favara.

2. Purified Gβγ dimer (69 mg/mL) [23] comprising human Gβ₁ and Gγ₂ (incorporating a C68S mutation to prevent post-translational lipidation). Expression and purification steps are detailed in the supplementary data section of [25]. Store at -80 °C. Available on request from Dr David Favara.

Reagents

1. Roche cOmplete, EDTA-free protease inhibitor cocktail tablets (Roche, catalog number: 11873580001); store at 4 °C

2. Phosphate-buffered saline (PBS), pH 7.4, 500 mL (Gibco, catalog number: 10010015)

3. Fetal bovine serum (FBS) tetracycline-free, 500 mL (Biosera, catalog number: FB-1001T/500); store at -20 °C

4. Trypan Blue solution, 0.4% (Gibco, catalog number: 15250061)

5. Anti-HA monoclonal antibody conjugated to APC, 200 μL (Miltenyi Biotec, catalog number: 130-123-553); store at 4 °C

6. HEPES buffer (molecular biology grade, ≥99%), 1 kg (Fisher BioReagents, catalog number: BP3101-1)

7. 4 N NaOH, 4 L (Supelco, catalog number: 1115845000)

8. 1 M NaOH, 500 mL (Supelco, catalog number: 79724-500ML)

9. PES membrane filter, 0.22 μm pore size (Millipore, catalog number: GPWP04700)

10. NaCl (Molecular biology grade, ≥99%), 500 g (Sigma-Aldrich, catalog number: S3014-500G)

11. MgCl₂ hexahydrate (molecular biology grade, ≥99%), 500 g (Fisher BioReagents, catalog number: BP214500)

12. Glycerol (molecular biology grade, ≥99%), 1 L (Sigma-Aldrich, catalog number: G5516-1L)

13. Tris-(2-Carboxyethyl)phosphine, hydrochloride (TCEP) (molecular biology grade, ≥98%), 1 g (Sigma-Aldrich, catalog number: 75259-1G)

14. Apyrase (500 units/mL), 50 units (NEB, catalog number: M0398L); store at -20 °C

15. Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, 5 g (Thermo Scientific, catalog number: 36978)

16. Isopropanol (molecular biology grade, 99.5%), 1 L (Acros, catalog number: 10215331)

17. Lauryl maltose neopentyl glycol (LMNG), 25 g (Anatrace, catalog number: NG310 25 GM); store at -20 °C

18. Cholesteryl hemisuccinate tris salt (CHS), 5 g (Sigma-Aldrich, catalog number: C6013-5G); store at -20 °C

19. Benzonase nuclease, 25 KU (Millipore, catalog number: E1014-25KU); store at -20 °C

20. Standard SDS-PAGE reagents and pre-stained protein ladder (any suitable supplier)

21. Iba Lifesciences Strep-Tactin XT 4Flow high-capacity resin, 50 mL, 50% suspension (Iba Lifesciences, catalog number: 2-5030-025); store at 4 °C

22. Iba Lifesciences biotin, 5 g (Iba Lifesciences, catalog number: 2-1016-005); store at 4 °C

23. Iba Lifesciences buffer XT-R, 250 mL (Iba Lifesciences, catalog number: 2-1045-250); store at 4 °C

24. Purified TEV protease (4.4 mg/mL); expression and purification steps detailed in [29]. Alternatively, this can be purchased from NEB (NEB, catalog number: P8112S)

Solutions

1. Flow cytometry buffer (see Recipes)

2. 3 M NaCl (see Recipes)

3. 1 M MgCl₂ (see Recipes)

4. 300 mM HEPES (see Recipes)

5. 50% glycerol (see Recipes)

6. 0.5 M TCEP (see Recipes)

7. 500 units/mL apyrase (see Recipes)

8. 0.5 M biotin (see Recipes)

9. 200 mM PMSF (see Recipes)

10. 5% LMNG (see Recipes)

11. 5% LMNG and 0.5% CHS (see Recipes)

12. Cell harvest buffer (see Recipes)

13. Solubilisation buffer (see Recipes)

14. Wash buffer (see Recipes)

15. Elution buffer (see Recipes)

16. SEC running buffer without glycerol (see Recipes)

17. SEC running buffer with 10% glycerol (see Recipes)

Recipes

1. Flow cytometry buffer

Note: Make up fresh and store at 4 °C. Keep on ice when in use.

2. 3 M NaCl

a. Weigh 175.32 g of NaCl and dissolve in 700 mL of ultrapure water.

b. Bring to a total volume of 1,000 mL with ultrapure water.

c. Filter through a 0.22 μm pore size PES membrane filter into a sterile bottle and store at room temperature.

3. 1 M MgCl₂

a. Weigh 20.33 g of MgCl₂ hexahydrate and dissolve in 50 mL of ultrapure water.

b. Bring to a total volume of 100 mL with ultrapure water. Store at room temperature.

4. 300 mM HEPES

a. Weigh 71.5 g of HEPES and dissolve in 900 mL of 4 °C ultrapure water. Keep on ice.

b. Measure pH and titrate carefully with 4 N NaOH (coarse titration) and then 1 M NaOH (fine titration) to pH 7.5 at 4 °C. Before titration, calibrate the pH meter with chilled buffers.

c. Bring to a total volume of 1,000 mL with ultrapure water.

d. Filter through a 0.22 μm pore size PES membrane filter into a sterile bottle and store at 4 °C.

5. 50% glycerol

a. Measure 500 mL of glycerol and mix with 500 mL of ultrapure water.

b. Autoclave at 121 °C for 15 min and store at 4 °C.

6. 0.5 M TCEP

a. Weigh 2.87 g of TCEP and dissolve it in 10 mL of ultrapure water.

b. Measure pH and titrate carefully with 4 N NaOH (coarse titration) and then 1 M NaOH (fine titration) to pH 7 at room temperature.

c. Bring to a total volume of 20 mL with ultrapure water.

d. Filter through a 0.22 μm pore size PES membrane filter into a sterile bottle.

e. Make up 1 mL aliquots and store at -20 °C.

Note: TCEP is a reducing agent that prevents the formation of non-native disulphide bonds. It is used to prevent deactivation of the TEV cysteine protease used for receptor cleavage.

7. 500 units/mL apyrase

Aliquoting for storage:

a. Source: apyrase 500 units/mL, 100 μL total, in 20 mM MES, 50 mM NaCl, 0.1 mM CaCl₂, 1 mM DTT, 0.1% Tween-20, 50% glycerol.

b. Dispense 10 μL into ten 0.2 mL PCR tubes. Each aliquot contains 5 units apyrase at 500 units/mL.

c. Store at -20 °C.

Note: Apyrase degrades nucleoside triphosphates (ATP, GTP) and diphosphates (ADP, GDP) to their corresponding monophosphates and inorganic phosphate. This enzymatic activity depletes nucleoside tri- or diphosphates that could otherwise destabilise the active-state ADGRL4-mini-G_q-βγ complex. Activity typically requires Ca²⁺ or Mg²⁺. In this protocol, we use 1 mM MgCl₂.

8. 0.5 M biotin

a. Place a glass beaker (≥50 mL) with a magnetic stir bar on a stir plate. Add 12 mL of ultrapure water and start stirring.

b. Weigh 5 g of biotin powder and add gradually while stirring to form a suspension. At this concentration, expect poor solubility below a pH of 7.5–8.

c. Measure pH and carefully titrate pH upward with 1 M NaOH in 0.5 mL increments. Switch to 100–200 μL increments of 1 M NaOH when the suspension becomes less thick and clearer. Allow time for each addition to fully mix before adding the next.

d. The suspension should become clear at pH 8–9.

e. Once suspension is completely clear, measure pH. The final target pH is between 8.2 and 8.4 If required, carefully titrate downward with 1 M HCl in 50–100 μL increments.

f. Once pH has settled at 8.2–8.4, bring to a final total volume of 40.94 mL with ultrapure water.

g. Filter through a 0.22 μm pore size PES membrane filter into a sterile bottle and store at 4 °C.

9. 200 mM PMSF

a. In a fume hood, weigh 3.484 g of PMSF into a beaker.

b. Add 80 mL of isopropanol and mix by swirling to completely dissolve all PMSF.

c. Once fully dissolved, bring to a total volume of 100 mL with additional isopropanol.

d. Aliquot into 10 mL portions in 15 mL tubes and store at -20 °C, protected from light.

Caution: PMSF is toxic and corrosive and should be prepared in a fume hood. Ensure that it is always carefully handled with gloves.

Note: Only add PMSF to aqueous buffers immediately before use due it being unstable in water.

10. 5% LMNG

a. In a clear beaker or 50 mL tube, weigh 1 g of LMNG, mix with 10 mL of ultrapure water, and mix gently until dissolved. Avoid vigorous shaking to minimise bubble formation.

b. Bring to a final volume of 20 mL with additional ultrapure water.

c. Filter through a 0.22 μm pore size PES membrane filter and aliquot into 1.5 mL microcentrifuge tubes.

d. Store at -20 °C.

11. 5% LMNG and 0.5% CHS

a. In a clear beaker or 50 mL tube, weigh 2 g of LMNG, mix with 20 mL of ultrapure water, and mix gently until dissolved. Avoid vigorous shaking to minimise bubble formation.

b. Weigh 0.2 g of CHS and add this to the LMNG solution.

c. Bring to a final volume of 40 mL with additional ultrapure water.

d. Gently mix overnight at 4 °C in a 50 mL tube using a tube roller.

e. Filter through a 0.22 μm pore size PES membrane filter into a sterile 50 mL tube.

f. Store at -20 °C.

12. Cell harvest buffer

Note: Make up fresh on the day of harvesting and keep chilled at 4 °C. Dissolving cOmplete EDTA-free protease inhibitor tablets will require a stir bar for approximately 10 min at 4 °C.

13. Solubilisation buffer

* Include the volume of the thawed cell pellet in your calculation. When snap-freezing cells in liquid nitrogen, make a note of the volume of the pellet in the 50 mL centrifuge tube. For 2 L of HEK293 GnTI^- TetR cells, this amounts to a ~25 mL volume.

Note: Make up fresh before use and keep on ice at 4 °C. Only add the benzonase and PMSF immediately before use. During solubilisation, 20 mL of 5% LMNG, 0.5% CHS is added to the above mixture, bringing the total volume to 100 mL (final detergent concentration being 1% LMNG, 0.1% CHS).

14. Wash buffer

Note: Make up fresh before use and keep on ice at 4 °C.

15. Elution buffer

Note: Make up fresh before use and keep on ice at 4 °C.

16. SEC running buffer without glycerol

Note: Make up fresh before use and keep on ice at 4 °C.

17. SEC running buffer with 10% glycerol

Note: Make up fresh before use and keep on ice at 4 °C.

Laboratory supplies

1. 96-well round (U) bottom plates (Thermo Scientific, catalog number: 163320)

2. 1 L polypropylene bottle assembly (Beckman Coulter, catalog number: C31597)

3. 50 mL centrifuge tubes (Corning, catalog number: CLS430828-100EA)

4. Ultracentrifuge tubes: Beckman Coulter PC tube with aluminium cap, 26.3 mL volume (Beckman Coulter, catalog number: 355618)

5. Econo-column chromatography column, 2.5 × 10 cm (Bio-Rad, catalog number: 7372512)

6. Microcentrifuge tubes, 1.5 mL (Pierce, catalog number: 69715)

7. Amicon Ultra-15 centrifugal filter 100 kDa MWCO (15 mL sample volume) (Millipore, catalog number: UFC910024)

8. Amicon Ultra-4 centrifugal filter 100 kDa MWCO (4 mL sample volume) (Millipore, catalog number: UFC810024)

9. Amicon Ultra-0.5 centrifugal filter 100 kDa MWCO (0.5 mL sample volume) (Millipore, catalog number: UFC510096)

10. Direct Detect assay-free cards, 50 cards (Merck, catalog number: DDAC00010)

11. UltrAuFoil R1.2/1.3 300 mesh Au grids (Quantifoil, catalog number: Q350AR13A)

Equipment

1. Flow cytometer (Sony, model: ID7000 Spectral Cell Analyzer)

2. Temperature-controlled floor-standing centrifuge for 1 L bottles (Beckman Coulter, Avanti JXN-26 Refrigerated Centrifuge, model: JXN-26)

3. Ultrapure water system (Sartorius, model: Arium Pro Ultrapure Water System)

4. Beckman Coulter Type 70 Ti fixed-angle rotor (Beckman Coulter, catalog number: 337922)

5. Temperature-controlled floor-standing ultracentrifuge (Beckman Coulter Optima L-100XP Ultracentrifuge, model: L-100XP)

6. End-over-end rotator (Elmi Rotamix RM1, catalog number: ROTAMIX RM1)

7. Shimadzu HPLC system (Shimadzu Corporation) comprising the following components: Shimadzu degasser module (DGU-20A), Shimadzu solvent delivery module (LC-20AD), Shimadzu autosampler module (SIL-20AC), Shimadzu column oven module (CTO-20AC), Shimadzu UV-detector module (SPD-20A), Shimadzu fluorescence detector module (RF-20A), and Shimadzu fraction collector module (FRC-40)

8. HPLC guard column, Agilent Bio SEC-5 guard column (300 Å, 7.8 × 50 mm, 5 μm) (Agilent, catalog number: 5190-2530)

9. HPLC column, Agilent Bio SEC-5 column (300 Å 4.6 × 300 mm, 5 μm) (Agilent, catalog number: 5190-2528)

10. Temperature-controlled benchtop 1.5 mL tube centrifuge (Eppendorf, model: 5418 R)

11. Protein detection system, Direct Detect infrared spectrometer (Merck, catalog number: DDHW00010)

12. Automated vitrification system for cryo-EM (FEI, model: Vitrobot Mark IV)

13. Ethane cryostat and temperature controller (MiTeGen, catalog number: ML-TCCS-001); design and operation are described in [30]

14. Glow-discharge plasma system (Fischione, catalog number: Model 1070)

Procedure