Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-P-4EBP1 (T37/46) (Rabbit, polyclonal) | Cell Signalling | Cat#9459L; RRID:AB_330985 | WB (1:1000) |
| Antibody | Anti-4EBP1 (Rabbit, polyclonal) | Cell Signalling | Cat#9452S; RRID:AB_331692 | WB (1:1000) |
| Antibody | Anti-P-p70 S6 Kinase (T389) (Rabbit, monoclonal) | Cell Signalling | Cat#9205L; RRID:AB_330944 | WB (1:1000) |
| Antibody | Anti-Rabbit IgG, HRP-linked Antibody | Cell Signalling | Cat#7074; RRID:AB_2099233 | WB (1:5000) |
| Strain, strain background (Escherichia coli) | LOBSTR cells | KeraFast | Cat# EC1001 | Chemically competent cells |
| Cell line (Homo sapiens) | Expi293F cells | Thermo Fisher | Cat#A14527; RRID:CVCL_D615 | |
| Recombinant DNA reagent | pOPL24 (plasmid) | Anandapadamanaban et al., 2019 | 2xStrepII-1xFlag-Human_Raptor, in pCAG | |
| Recombinant DNA reagent | pOPL25 (plasmid) | Anandapadamanaban et al., 2019 | Human_mTOR nontagged, in pCAG | |
| Recombinant DNA reagent | pOPL26 (plasmid) | Anandapadamanaban et al., 2019 | 3xFlag-Human_LST8, in pCAG | |
| Recombinant DNA reagent | pOPL95 (plasmid) | This paper | Human_mLST8 nontagged, in pCAG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL119 (plasmid) | This paper | Human_Raptor nontagged, in pCAG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL121 (plasmid) | This paper | 2xStrepII(tev)-Human_mTOR, in pCAG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL146 (plasmid) | This paper | 2xStrepII(tev)-Human_mTOR_A1459P, in pCAG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL151 (plasmid) | This paper | 2xStrepII(tev)-Human_mTOR_1376–2549, in pCAG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL107 (plasmid) | This paper | GST(tev)-Human_mTOR_FRB_2015–2114, in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL40 (plasmid) | This paper | GST(tev)-Human_Rheb1, in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pMA9 (plasmid) |
Anandapadamanaban et al., 2019 This paper |
HisLIP(tev)-Human_PRAS40,in pOPTL (Available from RLW lab) |
|
| Recombinant DNA reagent | pAB87 (plasmid) | This paper | GST(PreScission)Human_DEPTOR_1–409 (S204, N389 natural variant) (Full length WT), in pGEX-6P1 (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL111 (plasmid) | This paper | GST(tev)-Human_DEPTOR_228_409 (Linker-PDZ), in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL127 (plasmid) | This paper | HisLIP(tev)-Human_DEPTOR_324_409 (PDZ), in pOPTL (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL138 (plasmid) | This paper | GST(tev)-Human_DEPTOR_1–409 (S204, N389 natural variant) (Full length WT), in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL139 (plasmid) | This paper | GST(tev)-Human_DEPTOR_1-409-13S/T-A mutant (T241A,S244A,T259A,S260A,S263A,S265A,S282A,S283A,S287A, S293A,S297A,S298A,S299A) (Full length 13A), in pOPTG (PCR-ed from Addgene clone 21702) (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL140 (plasmid) | This paper | GST(tev)-Human_DEPTOR_1–323 (DEP-DEP-linker), in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL141 (plasmid) | This paper | GST(tev)-Human_DEPTOR_1-323-13S/T-A mutant (DEP-DEP-linker 13A), in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL143 (plasmid) | This paper | GST(tev)-Human_DEPTOR_228-409-13S/T-A mutant (linker PDZ 13A), in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL157 (plasmid) | This paper | GST(tev)-Human_DEPTOR_1–220 (DEP-DEP), in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL159 (plasmid) | This paper | His(tev)-Human_DEPTOR_228–323 (Linker), in pOPTH(tev) (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL166 (plasmid) |
This paper | GST(tev)-Human_DEPTOR_305–409 (extended PDZ), in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL167 (plasmid) | This paper | GST(tev)-Human DEPTOR_315–409 (extended PDZ), in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOPL163 (plasmid) | This paper | GST(PreScission)-Human_S6K1αII,367–404, in pGEX-6P1 (Available from RLW lab) |
|
| Recombinant DNA reagent | pOP826 (plasmid) | This paper | in GST(tev)-Human 4EBP1, in pOPTG (Available from RLW lab) |
|
| Recombinant DNA reagent | pOP854 (plasmid) | This paper | GST(tev)-Human 4EBP1-TOS-less-(114-FEMDI-118/AAAAA), in pOPTG (Available from RLW lab) |
|
| Chemical compound, drug | NEBuilder HiFi assembly Master Mix | New England Biolabs | Cat#E2621S | |
| Chemical compound, drug | cOmplete EDTA-free protease inhibitor tablets | Roche | Cat#11873580001 | |
| Chemical compound, drug | Universal Nuclease | Pierce | Cat#88702 | |
| Chemical compound, drug | Desthiobiotin | IBA | Cat#2-1000-005 | |
| Chemical compound, drug | Glutathione Sepharose 4B resin | GE Healthcare | Cat#17-0756-05 | |
| Chemical compound, drug | Ni-NTA agarose resin | Qiagen | Cat#30230 | |
| Chemical compound, drug | Lysozyme | Sigma | Cat#L6876 | |
| Chemical compound, drug | TCEP (Tris(2-carboxyethyl) phosphine hydrochlorid) |
Soltec Ventures | Cat#M115 | |
| Chemical compound, drug | Bovine Serum Albumin | Thermo Fisher | Cat#BP1605 | |
| Chemical compound, drug | PEI (Polyethyleneimine‘MAX’, MW 40,000) | Polysciences | Cat#24765 | |
| Chemical compound, drug | SuperSignal West Pico PLUS chemiluminescent substrate | Thermo Fisher | Cat#34577 | |
| Chemical compound, drug | LDS sample buffer | NuPAGE | Cat#NP0008 | |
| Chemical compound, drug | Triton X-100 | Sigma | Cat#T8787 | |
| Chemical compound, drug | Tween 20 | NBS Biologicals | Cat#17767-B | |
| Chemical compound, drug | ATP | Jena Bioscience | Cat#NU-1010 | |
| Chemical compound, drug | GTPγS | Jena Bioscience | Cat#NU-412–20 | |
| Chemical compound, drug | Glutaraldehyde solution, 25% in water | Sigma | Cat#G5882 | |
| Chemical compound, drug | D2O | Acros Organics | Cat#351430075 | |
| Chemical compound, drug | InstantBlue Protein Stain | Expedeon | Cat#1SB1L | |
| Chemical compound, drug | Expi293 Expression Medium | Thermo Fisher | Cat#A1435102 | |
| Chemical compound, drug | Yeast Nitrogen Base Without Amino Acids and Ammonium Sulfate | Sigma | Cat#Y1251 | |
| Chemical compound, drug | Ammonium15N chloride | Sigma | Cat#299251 | |
| Chemical compound, drug | D-Glucose (U-13C699%) | Cambridge Isotopes Laboratores Inc | Cat#CLM-1396–10 | |
| Chemical compound, drug | 99.9% 2H atom D2O | Cortecnet | Cat#CD5251P1000 | |
| Software, algorithm | ASTRA software package for analysis of SEC-MALS data | Wyatt | http://www.wyatt.com/products/software/astra.html; RRID:SCR_016255 | |
| Software, algorithm | ProteinLynx Global Server | Waters | 720001408EN; RRID:SCR_016664 |
|
| Software, algorithm | DynamX | Waters | 720005145EN | |
| Software, algorithm | ImageJ | Schneider et al., 2012 | https://imagej.nih.gov/ij/; RRID:SCR_003070 | |
| Software, algorithm | GraphPad Prism | GraphPad Software |
https://www.graphpad.com/ RRID:SCR_002798 |
|
| Software, algorithm | Topspin | Bruker | https://www.bruker.com/products/mr/nmr/software/topspin.html; RRID:SCR_014227 | |
| Software, algorithm | NMRFAM-Sparky | Lee et al., 2015 |
http://pine.nmrfam.wisc.edu/download_packages.html RRID:SCR_014228 |
|
| Software, algorithm | MARS | Jung and Zweckstetter, 2004 | https://www3.mpibpc.mpg.de/groups/zweckstetter/_links/software_mars.html | |
| Software, algorithm | NMRPipe | Delaglio et al., 1995 | https://www.ibbr.umd.edu/nmrpipe/install.html | |
| Software, algorithm | MDD compressed sensing | Kazimierczuk and Orekhov, 2011 | http://mddnmr.spektrino.com/ | |
| Software, algorithm | POMONA | Shen and Bax, 2015 | https://spin.niddk.nih.gov/bax/nmrserver/pomona/ | |
| Software, algorithm | Rosetta and NMR restraints | Raman et al., 2010 | https://www.rosettacommons.org/software | |
| Software, algorithm | Random coil shifts | Kjaergaard and Poulsen, 2011; Kjaergaard and Poulsen, 2011; Schwarzinger et al., 2001. | https://www1.bio.ku.dk/english/research/bms/sbinlab/randomchemicalshifts1/ | |
| Software, algorithm | RELION three software package | Scheres, 2012 | http://www2.mrc-lmb.cam.ac.uk/relion; RRID:SCR_016274 | |
| Software, algorithm | Gctf | Zhang, 2016 | RELION three software package | |
| Software, algorithm | MotionCor2 | Zheng et al., 2017 | RELION three software package | |
| Software, algorithm | ResMap | Kucukelbir et al., 2014 | RELION three software package | |
| Software, algorithm | Chimera | Pettersen et al., 2004 |
http://plato.cgl.ucsf.edu/chimera/ RRID:SCR_004097 |
|
| Software, algorithm | Coot | Casañal et al., 2020; Emsley and Cowtan, 2004 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/; RRID:SCR_014222 | |
| Software, algorithm | ChimeraX | Pettersen et al., 2021 |
https://www.cgl.ucsf.edu/chimerax/ RRID:SCR_015872 |
|
| Software, algorithm | REFMAC5 | Brown et al., 2015; Murshudov et al., 1997 | https://www.ccp4.ac.uk; RRID:SCR_007255 | |
| Other | 0.45 μM Syringe Filter | Millipore Sigma | Cat#SE2M230I04 | |
| Other | 5 μM Minisart Syringe Filter | Sartorius | Cat#17594-Q | |
| Other | 5 mL StrepTrap HP column | GE Healthcare | Cat#28-9075-47 | |
| Other | 5 mL HiTrap Q HP column | GE Healthcare | Cat#17-1153-01 | |
| Other | 5 mL HiTrap Heparin HP column | GE Healthcare | Cat#17-0407-01 | |
| Other | 5 mL HisTrap FF column | GE Healthcare | Cat#17-5255-01 | |
| Other | HiLoad 16/60 Superdex 200 column | GE Healthcare | Cat#17-1069-01 | |
| Other | HiLoad 16/60 Superdex 75 column | GE Healthcare | Cat#17-1068-01 | |
| Other | Superose 6 increase10/300 GL | GE Healthcare | Cat#29-0915-96 | |
| Other | 4–12% BisTris NuPAGE Protein Gel | Thermo Fisher | Cat#NP0323 | |
| Other | SuperSep Phos-tag 7.5% precast gels | FUJIFILM Wako Chemicals | Cat#192–17381 | |
| Other | iBlot 0.2 µM pore size nitrocellulose Transfer Stacks | Thermo Fisher | Cat#IB301002 | |
| Other | 15 mL Amicon Ultra-153K Centrifugal Filters | Millipore Sigma | Cat#UFC900324 | |
| Other | 15 mL Amicon Ultra-1530K Centrifugal Filters | Millipore Sigma | Cat#UFC903024 | |
| Other | 15 mL Amicon Ultra-15100K Centrifugal Filters | Millipore Sigma | Cat#UFC910024 | |
| Other | 4 mL Amicon Ultra-4 100K Centrifugal Filters | Millipore Sigma | Cat#UFC810024 | |
| Other | Enzymate Pepsin Column | Waters | Cat#186007233 | |
| Other | Acquity UPLC BEH C18 VanGuard Pre-column | Waters | Cat#186003975 | |
| Other | Acquity UPLC BEH C18 column | Waters | Cat#186002346 |