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. Author manuscript; available in PMC: 2022 Jul 8.
Published in final edited form as: Cell. 2021 Jun 16;184(14):3689–3701.e22. doi: 10.1016/j.cell.2021.05.019

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Mouse monoclonal Anti-FLAG M2 clone Sigma-Aldrich F1804; RRID:AB_262044
Mouse monoclonal Anti-FLAG M2-Peroxidase Sigma-Aldrich A8592; RRID:AB_439702
Rabbit monoclonal Anti-Actin Sigma-Aldrich A2066; RRID:AB_476693
Rabbit monoclonal Anti-SREBP2 (McFarlane et al., 2015) IgG-22D5
Mouse monoclonal Anti-Scap L7 (Sakai et al., 1997) IgG-9D5
Mouse monoclonal Anti-Insig This paper IgG-17H1
Bacterial and Virus Strains
XL10-Gold Ultracompetent cells Agilent Cat# 200317
MAX Efficiency DH10Bac Competent cells ThermoFisher Cat# 10361012
Chemicals, Peptides, and Recombinant Proteins
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) Avanti Cat# 850457
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) Avanti Cat# 850757P
2-Hydroxypropyl-β-cyclodextrin (HPCD) Cyclodextrin Technologies Development Cat# THPB-P
25-Hydroxycholesterol Steraloids Cat# C6550–000
[3H]Cholesterol American Radiolabeled Chemicals Cat# ART 2133
[3H]25-hydroxycholesterol American Radiolabeled Chemicals Cat# ART 0766
[3H]Lanosterol American Radiolabeled Chemicals Cat# ART 0546A
3x-FLAG peptide APExBio Cat# A6001
5-Cholesten-3ß-OL Hemmisuccinate (CHS) Steraloids Cat# C6823–000; CAS 1510-21-0
Anti-FLAG M2 affinity resin Sigma-Aldrich Cat# A2220
Benzamidine hydrochloride monohydrate Goldbio Cat# B-050–250; CAS 1670-14-0
Benzonase EMDMillipore Cat# 70746–3
Bio-Beads SM-2 Absorbent Media BIO-RAD Cat# 1523920
Cholesterol Sigma Cat# C8667–5g
E64 Goldbio Cat# E-064–100; CAS 66701-25-5
EZ-Link NHS-LC-LC-Biotin ThermoFisher Cat# 21343
Fetal Calf Serum Sigma-Aldrich F2442–500ML
FLAG peptide Biomatik Custom order
FuGENE6 transfection reagent Promega Cat# E2691
Glyco-diosgenin (GDN) Anatrace Cat# GDN101;
CAS 1402423-29-3
Iodoacetamide Sigma-Aldrich Cat# I1149
Lanosterol Sigma Aldrich Cat# L5768
Lauryl Maltose Neopentyl Glycol (LMNG) Anatrace Cat# NG310;
CAS 1257852-96-2
Leupeptin hemisulfate Goldbio Cat# L-010–100; CAS 103476-89-7
mPEG maleimide, MW 5000 Nanocs Inc. Cat.# PG1-ML-5k-1
NEBuilder HiFI DNA Assembly NEB Cat# E2621L
Newborn calf lipoprotein-deficient serum (LPDS) (Goldstein et al., 1983) N/A
Ni-NTA agarose Qiagen Cat# 30250
Papain Worthington Cat# LS003119
Phenylmethylsulfonylfluoride (PMSF) Goldbio Cat# P-470–25;
CAS 329-98-6
Protease Inhibitor Cocktail Set III, EDTA-Free Calbiochem Cat# 539134
Protein L agarose Pierce Cat# 20510
Sodium butyrate Sigma-Aldrich Cat# 303410;
CAS 156-54-7
Sodium cholate Sigma-Aldrich Cat# C1254;
CAS 206986-87-0
Sodium compactin (Brown et al., 1978) N/A
Sodium mevalonate (Brown et al., 1978) N/A
Sodium oleate (Goldstein et al., 1983) N/A
Tris (2-Carboxyethyl) phosphine Hydrochloride (TCEP- HCl) Goldbio CAT# TCEP1
CAS 51805- 45-9
X-tremeGENE HP DNA transfection reagent Millipore Sigma Cat# XTGHP-RO
Critical Commercial Assays
RNEAsy Mini kit Qiagen Cat# 74106
Superscript III First Strand kit Invitrogen Cat# 18080–51
GoTaq Polymerase kit Promega M3005
Deposited Data
PDB model of cScap L1-L7/4G10Fab This paper PDB 7LKF
PDB model of cScapD435V L1-L7/4G10Fab This paper PDB 7LKH
Focused refinement map of cScap L1-L7/4G10Fab This paper EMD-23405
Focused refinement map of cScapD435V L1-L7/4G10Fab This paper EMD-23408
Global map of cScap L1-L7/4G10Fab This paper EMD-23406
Global map of cScap L1-L7/4G10Fab This paper EMD-23407
PDB for starting template of Fab (Ebenhoch et al., 2019) PDB 6SVL
PDB for starting template of Fab (Hennen et al., 2016) PDB 5E94
PDB template for Scap TM homology model (Li et al., 2016b) PDB 5I31
PDB template for Insig homology model (Ren et al., 2015) PDB 4XU4
Experimental Models: Cell Lines
Human: HEK 293s GnTI− cells ATCC CRL-3022
Human: HEK 293T cells ATCC CRL-3216
Hamster: Scap-deficient SRD-13A cells (Rawson et al., 1999) N/A
Hamster: CHO K1 ATCC CCL-61
Spodoptera frugiperda: Sf9 cells Invitrogen Cat# 11496015
Oligonucleotides
Primers for Fab sequencing are listed in Table S3 This paper N/A
Recombinant DNA Plasmids
pEZT-Chicken Scap (D435V)-3xFLAG This paper N/A
pEZT-Chicken Scap (WT)-3xFLAG This paper N/A
pEZT-Chicken Scap (R135E D136K)-3xFLAG This paper N/A
pEZT-His8-Chicken Scap-3xFLAG D435V This paper N/A
pEZT-His8-Chicken Scap-3xFLAG WT This paper N/A
pEZT-Chicken Insig-1 This paper N/A
pEZT-Chicken Insig-1–2A-Scap (D435V)-3xFLAG This paper N/A
pEZT-Human Scap WT This paper N/A
pEZT-Myc-Human SREBP2 (CR) (D468A, S480A, R481S, and R519A) This paper N/A
pTK-HSV-Human SREBP2 (Hua et al., 1996b) N/A
pTK-Chicken Scap (D435V)-3xFLAG This paper N/A
pTK-Chicken Scap (WT)-3xFLAG This paper N/A
pTK-Hamster Scap (Hua et al., 1996a) N/A
pTK-Hamster Scap Y234A (Motamed et al., 2011) N/A
pTK-Hamster Scap Y640S (Zhang et al., 2013) N/A
pTK-Hamster Scap C147A This paper N/A
pTK-Hamster Scap C169S This paper N/A
pTK-Hamster Scap C264A This paper N/A
pTK-Hamster Scap Q94E This paper N/A
pTK-Hamster Scap I96A This paper N/A
pTK-Hamster Scap L125W This paper N/A
pTK-Hamster Scap R135W This paper N/A
pTK-Hamster Scap V150W This paper N/A
pTK-Hamster Scap K246W This paper N/A
pTK-Hamster Scap L636W This paper N/A
pTK-Hamster Scap I655A This paper N/A
pFastBac His10 Hamster(L1-L7) (Zhang et al., 2016) N/A
pFastBac His10 Hamster(L1-L7) C147A This paper N/A
pFastBac His10 Hamster(L1-L7) C169S This paper N/A
pFastBac His10 Hamster(L1-L7) C246A This paper N/A
pFastBac-Chicken (L1-L7)-His10 This paper N/A
pFastBac-His10-Chicken Scap CTD This paper N/A
pFastBac-3xFLAG-Human SREBP2 CTD (Kober et al., 2020) N/A
Software and Algorithms
PyMOL (2.3.5) Schrödinger https://pymol.org/2/
UCSF Chimera (Pettersen et al., 2004) https://www.cgl.ucsf.edu/chimera/
ISOLDE (Croll, 2018) https://isolde.cimr.cam.ac.uk/
SWISS-MODEL (Waterhouse et al., 2018) https://swissmodel.expasy.org/
Relion 3.1 (Zivanov et al., 2018) https://www3.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page
Phenix (1.18.2–3874) (Liebschner et al., 2019) https://www.phenix-online.org/download/
DALI server (Holm, 2020) http://ekhidna2.biocenter.helsinki.fi/dali/
PROMALS3D server (Pei et al., 2008) http://prodata.swmed.edu/promals3d/promals3d.php
Coot (Emsley et al., 2010) https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
IMGT database (Giudicelli et al., 2011) http://www.imgt.org/
Prism 8 GraphPad https://www.graphpad.com/scientific-software/prism/