Table S1. Content List of Extraction Buffers for Gpcrs

Table S1. Content list of extraction buffers for GPCRs. Receptor Detergent Salt pH buffer Ligand Additives rhodopsin1F88 0.5-0.65% HPTO, 90-120 mM 30-50 mM MES, pH 6.3-6.4 N/A N/A NG/ protein ratio ~2.2(w/w) Zn-acetate Na-acetate, pH 6.0 2RH1 10 mg/ml benzamidine, β2AR 1% DDM 500 mM NaCl 20 mM Tris, pH 7.4 N/A 10 mg/ml leupeptin, 200 μmol PMSF native 1% OG N/A N/A N/A N/A opsin3CAP 2VT4 β1AR 0.5% DDM 350 mM NaCl 20 mM Tris-HCl, pH 8 N/A 1 mM EDTA 3EML A2AAR 0.5% (w/v) DDM, 0.1% (w/v) CHS 10 mM MgCl2 10 mM HEPES, pH 7.5 4 mM theophylline 2 mg/mL iodoacetamide, PIC 20 mM KCl D3R3PBL 1% (w/v) DDM, 0.2% (w/v) CHS 10 mM MgCl2 10 mM HEPES, pH 7.5 200 μM eticlopride 2 mg/ml iodoacetamide, PIC 20 mM KCl CXCR43ODU 0.5% (w/v) DDM, 0.1% (w/v) CHS 500 mM NaCl 50 mM HEPES, pH 7.5 100 μM IT1t PIC 2Y00 β1AR 0.5% DDM 350 mM NaCl 20 mM Tris-HCl, pH 8 N/A 1 mM EDTA 3QAK A2AAR 0.5% (w/v) DDM, 0.1% (w/v) CHS 10 mM MgCl2 10 mM HEPES, pH 7.5 N/A 2 mg/ml iodoacetamide, PIC 20 mM KCl 3RZE 20% (v/v) glycerol, H1R 1% (w/v) DDM, 0.2% (w/v) CHS 500 mM NaCl 20 mM HEPES, pH 7.5 5 mM doxepin 10 mg/ml iodoacetamide, PIC 3SN6 10 mg/ml benzamidine, β2AR 1% DDM 500 mM NaCl 20 mM Tris, pH 7.4 N/A 10 mg/ml leupeptin, 200 μM PMSF 3V2Y 10 mM MgCl2 2 mg/ml iodoacetamide, S1P1R 0.5% (w/v) DDM, 0.1% (w/v) CHS 10 mM HEPES, pH 7.5 250 μM ML056 20 mM KCl 40% glycerol, PIC M3R4DAJ 1% DDM, 0.03% CHS 750 mM NaCl 30 mM HEPES, pH 7.5 1 μM titropium 30% glycerol, bromide 2 mg/ml iodoacetamide M2R3UON digitonin/Na-cholate 50 mM NaCl 10 mM KPB, pH 7.0 N/A 1 mM EDTA, PIC κ-OR4DJH 1% (w/v) DDM, 0.2% (w/v) CHS 150 mM NaCl 50 mM HEPES, pH 7.5 20 μM naltrexone 2 mg/ml iodoacetamide, PIC μ-OR4DKL 0.5% DDM, 0.3% CHAPS, 0.03% 500 mM NaCl 20 mM HEPES, pH 7.5 1 μM naloxone 30% (v/v) glycerol, CHS 2 mg/ml iodoacetamide, 100 μM TCEP 4EA3 500 mM NaCl 5% (v/v) glycerol, NOP 0.5% (w/v) DDM, 0.1% (w/v) CHS 50 mM HEPES, pH 7.5 25 μM C-24 20 mM KCl 1 mg/ml iodoacetamide δ-OR4EJ4 1.0% LMNG, 0.3% Na-cholate, 500 mM NaCl 20 mM HEPES, pH 7.5 1 μM naltrindole 30% (v/v) glycerol, 0.03% CHS 2 mg/ml iodoacetamide NTSR14GRV 1% LMNG/0.1% CHS 500 mM NaCl 50 mM Tris-HCl, pH 7.4 10 μM NTS 30% glycerol PAR13VW7 1% DDM, 0.03% CHS, 350 mM NaCl 20 mM Tris-HCl, pH 7.5 100 nM vorapaxar 10% glycerol, 0.2% Na-cholate 2 mg/ml iodoacetamide 4IAR 5-HT1B 1% (w/v) DDM, 0.2% (w/v) CHS 200 mM NaCl 50 mM HEPES, pH 7.5 50 μM ERG 2 mg/ml iodoacetamide, PIC SMO4JKV 1% (w/v) DDM, 0.2% (w/v) CHS 200 mM NaCl 50 mM HEPES, pH 7.5 30 μM LY2940680 2 mg/ml iodoacetamide, PIC 4K5Y CRF1R 2% (w/v) DM 500 mM NaCL 50 mM Tris-HCl, pH 8.0 0.3 μM CP-376395 N/A GCGR4L6R 1% (w/v) DDM, 0.1% (w/v) CHS 166 mM NaCl 25 mM HEPES, pH 7.0 270 μM NNC0640 13.3% glycerol, PIC CCR54MBS 0.5% (w/v) DDM, 0.1% (w/v) CHS 150 mM NaCl 50 mM HEPES, pH 7.5 100 μM Maraviroc 2 mg/ml iodoacetamide, PIC 4IB4 5-HT2B 1% (w/v) DDM, 0.2% (w/v) CHS 150 mM NaCl 10 mM HEPES, pH 7.5 50 μM ERG PIC 4GPO β1AR 2% DDM 350 mM NaCl 20 mM Tris-HCl, pH 8 N/A PIC 4MQS 30% glycerol, M2R 1% DDM, 0.03% CHS 750 mM NaCl 30 mM HEPES, pH 7.5 1 μM atropine 2 mg/ml iodoacetamide δ-OR4N6H 0.75% (w/v) DDM, 0.15% (w/v) CHS 500 mM NaCl 50 mM HEPES, pH 7.5 25 μM naltrindole N/A 4OR2 mGluR1 1% (w/v) DDM, 0.2% (w/v) CHS 200 mM NaCl 50 mM HEPES, pH 7.5 15 μM FITM 2 mg/ml iodoacetamide, PIC 4NTJ 1 M NaCl P2Y12R 0.5% (w/v) DDM, 0.1% (w/v) CHS 50 mM HEPES, pH 7.5 200 μM AZD1283 2 mg/ml iodoacetamide, PIC 10 mM MgCl2 20 mM KCl 4PXZ 1 M NaCl P2Y12R 0.5% (w/v) DDM, 0.1% (w/v) CHS 50 mM HEPES, pH 7.5 20 μM 2MeSADP or 2 mg/ml iodoacetamide, PIC 10 mM MgCl2 2MeSATP 20 mM KCl 4OO9 mGluR5 1.5% (w/v) DDM 250 mM NaCl 40 mM HEPES, pH 7.5 40 μM mavoglurant 8 mM iodoacetamide, PIC GPR404PHU 1% (w/v) DDM, 0.1% (w/v) CHS 500 mM NaCl 20 mM HEPES, pH 7.6 1 μM TAK-875 10 mM imidazole, PIC, 1 mg/ml iodoacetamide SMO4QIN 1% (w/v) DDM, 0.2% (w/v) CHS 200 mM NaCl 50 mM HEPES, pH 7.5 15 μM SAG1.5 PIC, 1 mg/ml iodoacetamide US284XT1 1% (w/v) DDM, 0.2% (w/v) CHS 150 mM NaCl 10 mM HEPES, pH 7.4 N/A 20% (v/v) glycerol, 2 mg/ml iodoacetamide 4S0V 1% (w/v) DDM, 0.2% Na-cholate, 10% glycerol, OX2R 500 mM NaCl 50 mM Tris, pH 7.5 5 μM suvorexant 0.2% CHS 2 mg/ml iodoacetamide 4XNV 500 μM ATP, PIC, 2 mg/ml P2Y1R 0.5% (w/v) DDM, 0.1% (w/v) CHS 300 mM NaCl 50 mM HEPES, pH 7.5 N/A iodoacetamide 4YAY AT1R 1% (w/v) DDM, 0.2% (w/v) CHS N/A N/A N/A N/A rhodopsin4ZWJ 0.5% (w/v) DDM, 0.1% (w/v) CHS 100 mM NaCl 20 mM Tris-HCl, pH 7.4 N/A 10% glycerol, PIC μ-OR5C1M 0.5% DDM, 0.3% CHAPS, 500 mM NaCl 20 mM HEPES, pH 7.5 1 μM naloxone 30% (v/v) glycerol, 0.03% CHS 2 mg/ml iodoacetamide, 100 μM TCEP 4ZJ8 1% (w/v) DDM, 0.2% Na-cholate, 10% glycerol, OX1R 500 mM NaCl 50 mM Tris-HCl, pH 7.4 5 μM SB-674042 0.2% (w/v) CHS 2 mg/ml iodoacetamide M1R5CXV 1% DDM, 0.03% CHS 750 mM NaCl 30 mM HEPES, pH 7.5 1 μM titropium 30% glycerol bromide 1 μM atropine M4R5DSG 1% DDM, 0.03% CHS 750 mM NaCl 30 mM HEPES, pH 7.5 1 μM titropium 30% glycerol bromide 1 μM atropine Table S2. List of crystallization conditions. Receptor Concentration Detergent Method Temperature Time Drop Precipitant Size (mg/ml) (w/v) (oC) (nl) (nl) (μm) rhodopsin1F88 7-9 0.55-0.75% HPTO + 0.4-0.55% NG HDVD 3-5 3 months N/A N/A 100×10×50 2RH1 β2AR 50 0.1% DDM LCP 21-23 N/A 25 or 800 30×15×5 50 native 5.0 1% OG HDVD 4 7 days 2,000 2,000 100×100×200 opsin3CAP 2VT4 β1AR 6.0 0.02% DM HDVD 18 N/A equal volume 240×40×10 3EML A2AAR 70 0.05% DDM + 0.001% CHS LCP 20 N/A 50 800 100×10×5 D3R3PBL 20-30 0.05% DDM + 0.01% CHS LCP 20 N/A 40-50 800 45×30×10 CXCR43ODU 60-70 0.05% DDM + 0.01% CHS LCP 20 10 days 40-50 800 90×40×5 2Y00 β1AR 14.5 0.45-1.8 mg/ml CHS + 0.5-0.65% SDVD 4 NA 200 N/A N/A Hega-10 3QAK A2AAR 60 0.025% DDM + 0.005% CHS LCP 20 7 days 40 800 150×20×5 3RZE H1R 30-40 0.025% DDM+0.005% CHS LCP 20 N/A 40-50 800 25-30 3SN6 β2AR 50 0.1% DDM (β2AR-agonist) LCP 20 3-4 days 40 800 250 0.02% LMNG (β2AR-Gs) 3V2Y 14 (1 day) S1P1R 100 0.05% DDM + 0.001% CHS LCP 3 days 25 800 50×10×5 17 (2 days) M3R4DAJ 60 0.01% LMNG + 0.001% CHS LCP N/A 2-3 days 15 600 20 M2R3UON 50 1% LMNG LCP 20 3-4 days 40 800 N/A κ-OR4DJH 40 0.03% DDM + 0.006% CHS LCP 20 N/A 45 800 60×20×10 μ-OR4DKL 30 0.01% LMNG + 0.001% CHS LCP N/A 5 days 80 700 20 NOP4EA3 40 0.025% DDM + 0.005% CHS LCP 20 14 days 40 800 40×10×3 δ-OR4EJ4 50 0.01% LMNG + 0.001% CHS LCP N/A 5 days 20-55 700 10-20 NTSR14GRV 20-25 0.003% LMNG + 0.0003%CHS LCP N/A 7 days 65 750 N/A PAR13VW7 40-50 0.1% DDM + 0.02% CHS LCP N/A 7 days 20-40 700 20 4IAR 5-HT1B 50-80 0.05% DDM + 0.01% CHS LCP 20 2-3 days 40 800 20-50 SMO4JKV 50-60 0.03% DDM + 0.006% CHS LCP 20 2-3 days 40 800 100×20×20 4K5Y CRF1R 20-30 0.15% DM LCP 22.5 N/A 40-60 750 20-30 GCGR4L6R 80 0.02% DDM + 0.004% CHS LCP 20 5 days N/A N/A 50–100 CCR54MBS 40-50 0.05% DDM + 0.01% CHS LCP 20 14 days 45 800 100×20×15 4IB4 5-HT2B 50 0.05% DDM + 0.01% CHS LCP 20 3 days 40 800 100×30×20 4GPO β1AR 8 0.02% DDM HDVD 18 N/A 1,000 1,000 N/A M2R4MQS N/A 0.01% LMNG + 0.001% CHS LCP N/A 2 days 30- 600 N/A 100 δ-OR4N6H 20 0.02% DDM + 0.004% CHS LCP 20 10 days 40 800 50×10×3 4OR2 mGluR1 50-80 0.05 % DDM + 0.01% CHS LCP 20 5-6 days N/A N/A 35×35×15 4NTJ P2Y12R ~40 0.05% DDM + 0.01% CHS LCP N/A 2 weeks 40 800 150×15×15 4PXZ P2Y12R 30 0.05% DDM + 0.01% CHS LCP N/A 2 weeks 40 800 80×50 ×5 4OO9 mGluR5 ~25 0.03% DDM LCP 20 N/A 40-60 750 40 GPR404PHU 25-35 0.02% LMNG + 0.0003% CHS LCP 20 1-2 days 100 800 25 SMO4QIN 50-60 0.03% DDM + 0.006% CHS LCP 20 N/A 50 1,000 N/A US284XT1 34 0.02% DDM + 0.004% CHS LCP 20 3-5 days 30 700 20 4S0V OX2R 30 0.05% LMNG + 0.02% CHS LCP 20 3 days 40 800 20 4XNV P2Y1R N/A 0.05% DDM + 0.01% CHS LCP N/A 2 weeks 40-50 800 100-130 4YAY AT1R 30 N/A LCP 20 6.5 hours SFX (65,000) 10×2×2 rhodopsin4ZWJ 40-50 0.005% LMNG + 0.001% CHS LCP 20 4 days 50 800 5-10 μ-OR5C1M 50 0.01% LMNG + 0.001% CHS LCP N/A 7 days 30 500 N/A 4ZJ8 OX1R >30 0.05% LMNG LCP 20 7 days 40 800 40 M1R5CXV 50 0.01% LMNG + 0.001% CHS LCP 20 24 hours 50 800 N/A M4R5DSG 50 0.01% LMNG + 0.001% CHS LCP 20 24 hours 20-40 600 N/A Table S3. Content list of precipitants for crystallization. Receptor PEG (v/v) Salt pH buffer Ligand Additives rhodopsin1F88 N/A 65-90 mM ZnOAc 30-50 mM MES, pH 6.3-6.4 N/A 5-7 mM β-mercaptoethanol,

840-860 mM (NH4)2SO4 NaOAc, pH 6.0 0.55-0.75% HPTO, 0.45-0.55% NG 2RH1 β2AR 30-35% PEG400 100-200 mM Na2SO4 100 mM Bis-Tris propane, pH 6.5-7.0 N/A 5-7% (v/v) 1,4-butanediol native opsin3CAP N/A N/A N/A N/A N/A 2VT4 β1AR 29-32% PEG600 N/A 100 mM ADA:NaOH, pH 6.9-7.3 N/A N/A 3EML A2AAR 30% PEG400 186 mM Li2SO4 100 mM Na citrate, pH 6.5 200 μM ZM241385 N/A 3PBL D3R 26-34% PEG400 200-350 mM NH4OAc 100 mM Bis-Tris propane, pH 7.5-8.0 1 mM eticlopride 2-3% (w/v) D-(+)-Glucose CXCR43ODU 20% PEG400 300 mM Na malonate 100 mM Na citrate, pH 5.5 2 mM IT1t 5 mM Taurine 2Y00 β1AR 25% PEG600 N/A 100 mM Tris-HCl, pH 8.5 N/A N/A 100 mM Bicine, pH 9.0

3QAK A2AAR 30% PEG400 150-200 mM MgCl2 100 mM Na citrate, pH 5.0-5.5 N/A N/A

3RZE H1R 26-30% PEG400 300 mM (NH4)3PO4, 100 mM Na citrate, pH 4.5 1 mM doxepin N/A

10 mM MgCl2 3SN6 β2AR 18-22% PEG400 350-450 mM KNO3 100 mM MES, pH 6.5 10 μM BI-167107 1 mM TCEP, 10 mM foscarnet

3V2Y S1P1R 34-36% PEG400 80 mM Na citrate 100 mM Tricine, pH 7.75 N/A 4% (v/v) glycerol 4DAJ M3R 27-38% PEG300 100 mM (NH4)3PO4 100 mM HEPES, pH 7.5 N/A 1% (w/w) 1,2,3-heptanetriol 3UON M2R 25-35% PEG300 100 mM (NH4)3PO4 100 mM HEPES, pH 7.0-7.8 N/A 2% 2-methyl-2,4- 4DJH pentanediol κ-OR 28-32% PEG400 350-450 mM KNO3 100 mM Na citrate, pH 5.8-6.4 N/A N/A 4DKL μ-OR 30-38% PEG400 300 mM Li2SO4 100 mM HEPES, pH 7.0 N/A 7.5% DMSO NOP4EA3 25-30% PEG400 100-200 mM KNa 100 mM Bis-Tris propane, pH 6.4 N/A N/A tartrate δ-OR4EJ4 29-33% PEG400 120-180 mM Na citrate/ 100 mM HEPES, pH 7.5 N/A N/A

350 mM MgCl2 NTSR14GRV 20.8% PEG400 43 mM KNa tartrate 80 mM HEPES, pH 7.0 N/A 2 mM TCEP 3VW7 PAR1 25-35% PEG300 100-200 mM NaCl 100 mM Na3PO4, pH 6.0-6.5 N/A N/A 4IAR 5-HT1B 30% PEG400 400 mM LiCl 100 mM Tris-HCl, pH 7.5 N/A N/A 4JKV SMO 32% PEG400 70 mM NH4F 100 mM HEPES, pH 7.8 N/A 4-8% (v/v) polypropylene glycol P 400 4K5Y CRF1R 30% PEG400 200 mM Li2SO4 100 mM Na citrate, pH 5.5 0.6 μM CP-376395 N/A GCGR4L6R 9-17% PEG400 140-200 mM KNa 100 mM MES, pH 6.0 200 μM NNC0640 0.35-0.55% (v/v) tartrate Jeffamine M-600 CCR54MBS 32-38% PEG400 100-200 mM NaCl 100 mM HEPES, pH 7.0 1 mM Maraviroc N/A 4IB4 5-HT2B 30% PEG400 50-75 mM MgSO4 100 mM Tris-HCl, pH 8.0-8.5 N/A N/A 4GPO β1AR 26-30% PEG200 100-300 mM (NH4)2SO4 20 mM Na citrate, pH 3.6-4 N/A N/A M2R4MQS 10-20% PEG300 N/A 100 mM HEPES, pH 7.2-7.9 N/A 1.2% 1,2,3-heptanetriol, 20-80 mM EDTA δ-OR4N6H 31-34% PEG400 95-120 mM KNa tartrate 100 mM MES, pH 6.1-6.2 1 mM naltrindole 5% (v/v) ethylene glycol 4OR2 mGluR1 27-30% PEG400 80-120 mM (NH4)3HPO4 100 mM HEPES, pH 7.0 N/A 4-8 mM TCEP 4NTJ P2Y12R 35% PEG400 50-150 mM NH4HCO2 100 mM Na cacodylate, pH 6.0-6.5 200 μM AZD1283 N/A 4PXZ P2Y12R 30-40% PEG400 300-450 mM NH4OAc 100 mM Na citrate, pH 5.0 500 μM 2MeSADP 3% v/v 1-propanol 4OO9 mGluR5 24-34% PEG400 100-250 mM 100 mM MES, pH 5.5-6.8 50 μM mavoglurant N/A

(NH4)3HPO4 GPR404PHU 29-31% 200 mM Na malonate/ 100 mM Tris-HCl, pH 8.0/ N/A N/A

PEG400/ 100 mM (NH4)3PO4 100 mM Bis-Tris propane, pH 7.2 39.8% PEG400 4QIN SMO 30% PEG400 100 mM MgSO4 100 mM MES, pH 6.0 N/A 2-3% polypropylene glycol US284XT1 32% PEG300 100 mM Na succinate, 100 mM MES, pH 6.3 N/A 1%P 400 (v/v) polypropylene pH 7.0 glycol P 400 4S0V OX2R 31% PEG400 200 mM Na formate 100 mM Na citrate, pH 5.9 N/A 3% 2,5-hexanediol 4XNV P2Y1R 0-10% PEG2000 100-300 mM 100 mM Na citrate, pH 6.5 50 μM BPTU N/A

MME (NH4)3HPO4 4YAY AT1R 28% PEG400 450 mM (NH4)3PO4 100 mM Na citrate, pH 5.0 N/A 4% (v/v) DMSO rhodopsin4ZWJ 28% PEG400 50 mM MgOAc 50 mM Na citrate, pH 5.0 N/A N/A μ-OR5C1M 15-25% PEG300 N/A 100 mM HEPES, pH 7.0-7.5 N/A 1% 1,2,3-heptanetriol, 0.5-1.0% polypropylene glycol P 400 4ZJ8 OX1R 32% PEG400 200 mM HCO₂K 100 mM Na citrate, pH 5.6 N/A 4% 1,4-butanediol M1R5CXV 33% PEG300 100 mM NaOAc 100 mM Bis-Tris propane, pH 8.0 N/A N/A M4R5DSG 25-40% PEG300 N/A 100 mM MES, pH 5.5-6.5 N/A 50-100 mM EDTA, pH 8.0