REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-CD3ε clone UCHT-1	Hybridoma	PMID 6788570
Anti-CD3ε clone UCHT-1 FITC conjugated	Biolegend	Cat# 300452
Anti-CD3ε clone UCHT-1 PE conjugated	Biolegend	Cat# 300408
Anti-CD8 clone SK1 PE-Cy7 conjugated	Biolegend	Cat# 344750
Anti-CD69 clone FN50 Pacific Blue conjugated	Biolegend	Cat# 310920
Bacterial and virus strains
Rosetta 2 (DE3) pLysS	Novagen	Cat# 71404-3
One Shot Top10 chemically competent E. coli	Invitrogen	Cat# C404010
Chemicals, peptides, and recombinant proteins
Alexa Fluor™ 647 Antibody Labelling Kit	Invitrogen	Cat# A20186
Alexa Fluor™ 555 Antibody Labelling Kit	Invitrogen	Cat# A20187
Amicon Ultra-15 centrifugal filters	Merck, Millipore	Cat# UFC901096
Ampicillin	Sigma	Cat# A9518-25G
L-Arginine	Sigma	Cat# A5131-1KG
BD Quantibrite™ Beads PE Fluorescence Quantification Kit	BD Biosciences	Cat# 340495
Benzonase nuclease	Sigma	Cat# E1014-25KU
Calcium chloride, 1 M	Sigma	Cat# 21115-250ML
Chloramphenicol	Sigma	Cat# C0378-25G
Corning 500ml filter system, 0.22 μM	Corning	Cat# 430758
Cystamine dihydrochloride	Sigma	Cat# C121509-25G
Cysteamine hydrochloride	Sigma	Cat# 30080-25G
Dialysis tubing cellulose membrane, 12 kDa	Sigma	D9402-100FT
DMEM medium (for growing CHO-K1 cells)	Gibco	Cat# 10938-025
DMEM medium (for growing HEK293T cells)	Sigma	Cat# D5976
DMSO	New England Biolabs	Cat# B0515A
DNA miniprep kit	Invitrogen	Cat# K210003
DNase I	Roche	Cat# 11284932001
1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (DGS-NTA(Ni2+) nickel salt)	Avanti Polar Lipids	Cat# 790404P-5mg
EDTA, 1 M, pH 8	Invitrogen	Cat# 15575-038
Express PES membrane filter Unit, 0.22 μm filter	Millipore	Cat# SLGP033RS
Filtropur S, 0.45 μm	Starsted	Cat# 83.1836
Filtropur V50, 500ml, 0.2 μm	Starsted	Cat# 83.3941.001
Fluo-4, AM, cell permeant	ThermoFisher	Cat# F14201
Foetal Bovine Serum	Gibco	Cat# 10500-064 lot. 08F6480K
Freestyle CHO expression medium (for growing CHO-S cells)	Gibco	Cat# 12651-014
GeneJuice transfection reagent 5x 1mL	Merck	Cat# 70967-6
L-glutamine, 200 mM	Sigma	Cat# G7513-100ML
Glyco-diosgenin (GDN)	Generon, Anatrace	Cat# GDN101-5GM
Hepes-NaOH, 1 M	Sigma	Cat# H0887-100ML
Hepes-NaOH pH 7.5, 1 M (TCR purification)	Gibco	Cat# 15630-056
Kanamycin	Sigma	Cat# K0254-20ML
LB agar	Sigma	Cat# L7025-500TAB
LB broth	Sigma	Cat# L7275-500TAB
Magnesium chloride, 1 M	Sigma	Cat# M1028-100ML
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)	Avanti Polar Lipids	Cat# 850457P-25mg
Penicillin (5,000 U)/Streptomycin (5 mg)/Neomycin (10 mg)	Sigma	Cat# P4083-100ML
Peptide, CTag: EDQVDPRLIDGK	Genscript	Custom Peptide
Peptide, gp100: YLEPGPVTV	Genscript	Custom Peptide
Peptide, 9V: SLLMWITQV	Genscript	Custom Peptide
Phosphate buffered saline	Oxoid	Cat# BR0014G
Protease inhibitors, cOmplete EDTA-Free	Roche	Cat# 11873580001 (5056489001)
RPMI Medium 1640	Gibco	Cat# 21875-034
Silver Stain Kit	Thermo Scientific, Pierce	Cat# 24612
Sodium chloride	Sigma	S9888-1KG
Sodium pyruvate, 100 mM	Sigma	Cat# S8636-100ML
Tris-HCl pH 8.0, 1 M	Sigma	Cat# T3038-1L
Triton-X-100, 100%	Sigma	Cat# T-9284
Trypsin solution from porcine pancreas	Sigma	Cat# T4549-100ML
Urea	Sigma	Cat# U1250-1KG
Deposited data
gp100/HLA-A2/TCR-CD3 complex structure	RCSB	PDB: 7PHR
gp100/HLA-A2/TCR-CD3 complex EM data	EMDB	EMD-13427
Experimental models: Cell lines
Hamster: CHO-K1	Lonza	N/A
Hamster: CHO-S	Invitrogen, Gibco	Cat# R80007
Human: HEK293T	ATCC	ATCC CRL-3216
Human: Jurkat	ATCC	ATCC TIB-152
Human: THP1	ATCC	ATCC TIB-202
Oligonucleotides
pEYFP reverse: ACCAGGATGGGCACCAC	IDT; Sigma	Lab ID: 554
pHR forward: TGCTTCTCGCTTCTGTTCG	IDT; Sigma	Lab ID: 1166
pHR reverse: CCACATAGCGTAAAAGGAGC	IDT: Sigma	Lab ID: 1167
pHRi forward: CAACAAGTTACCGAGAAAGAAGAACTCAC	IDT: Sigma	Lab pHRi-F-mHSP
T7 forward	Source Bioscience	In-house primers
T7 reverse	Source Bioscience	In-house primers
Recombinant DNA
pHRSin_hTCRαγεζ	Addgene	ID 187351
pHRSin_hTCRαγε	Addgene	ID 187352
pHRSin_hTCRβεζ	Addgene	ID 187353
pHRSin_hTCRβε	Addgene	ID 187354
pHRSin_hCD3δ-GFP2	Addgene	ID 187355
pET28a(+)_6xHIS-hHLA-A2 (MHC Class I heavy)-C-Tag	Addgene	ID 187356
pET28a(+)_beta2M (MHC Class I light)	Addgene	ID 187357
pHRSin-IRES-EmGFP_HPC-4 Ab heavy-6xHIS	Addgene	ID 187358
pHRSin-IRES-EmGFP_HPC-4 Ab light	Addgene	ID 187359
pHRSin_GPa3b17 TCRα	Addgene	ID 187360
pHRSin_GPa3b17 TCRβ	Addgene	ID 187361
pHRi_GPa3b17 TCRα	Addgene	ID 187601
pHRi_GPa3b17 TCRβ	Addgene	ID 187602
pHRSin_1G4 hTCRα	Addgene	ID 187572
pHRSin_1G4 hTCRβ-SNAP	Addgene	ID 187573
pHRi_1G4 hTCRα	Addgene	ID 187603
pHRi_1G4 hTCRβ-SNAP	Addgene	ID 187604
pMDG	Addgene	ID 187440
P8.91	Addgene	ID 187441
Software and algorithms
Calcium flux analysis code (custom)	In-house generate	https://github.com/janehumphrey/calcium
CCP4 program suite	Winn et al., 2011	RRID:SCR_007255
CHARMM-GUI	Jo et al., 2008; Lee et al., 2016	charmm-gui.org
COOT	Emsley and Cowtan, 2004	RRID:SCR_014222
cryoSPARC	Punjani et al., 2017	RRID:SCR_016501
Flowjo v10.7.1	N/A	https://www.flowjo.com
Gromacs v2020.6	Abraham et al., 2015	RRID:SCR_014565
Illustrate	Goodsell et al., 2019	github.com/ccsb-scripps/Illustrate
MDAnalysis v0.20.1	Gowers et al., 2016	github.com/MDAnalysis/mdanalysis/releases
NumPy v1.19.5	Harris et al., 2020	RRID:SCR_008633
OPM database	Lomize et al., 2012	RRID:SCR_011961
Phenix	Liebschner et al., 2019	RRID:SCR_014224
Prism v9.2.0	N/A	https://www.graphpad.com/scientific-software/prism/
PyMOL	The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC	RRID:SCR_000305
Rosetta	Conway et al., 2014	RRID:SCR_015701
RosettaCM	Song et al., 2013; Wang et al., 2016b	rosettacommons.org/docs/latest/application_documentation/structure_prediction/RosettaCM
Sc	Lawrence and Colman, 1993	https://www.ccp4.ac.uk/html/sc.html
Snapgene v4.1.9	N/A	https://www.snapgene.com
TOPAZ	Bepler et al., 2019	http://cb.csail.mit.edu/cb/topaz/
UCSF ChimeraX	Pettersen et al., 2021	RRID:SCR_015872
UCSF Chimera	Pettersen et al., 2004	RRID:SCR_004097
Visual Molecular Dynamics (VMD) v1.9.3	Humphrey et al., 1996	www.ks.uiuc.edu/Research/vmd/vmd-1.9.3/
Other
DNA sequencing	Source Bioscience	N/A