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- PDB-6m49: cryo-EM structure of Scap/Insig complex in the present of 25-hydr... -

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Basic information

Entry
Database: PDB / ID: 6m49
Titlecryo-EM structure of Scap/Insig complex in the present of 25-hydroxyl cholesterol.
Components
  • Insulin-induced gene 2 protein
  • Sterol regulatory element-binding protein cleavage-activating protein,Sterol regulatory element-binding protein cleavage-activating protein
KeywordsMEMBRANE PROTEIN / Scap / Insig / cholesterol / sterol sensing / 25-hydroxycholesterol / SREBP
Function / homology
Function and homology information


SREBP-SCAP complex retention in endoplasmic reticulum / SREBP-SCAP-Insig complex / cranial suture morphogenesis / SREBP-SCAP complex / negative regulation of steroid biosynthetic process / regulation of cholesterol biosynthetic process / sterol binding / SREBP signaling pathway / negative regulation of cholesterol biosynthetic process / COPII-coated vesicle cargo loading ...SREBP-SCAP complex retention in endoplasmic reticulum / SREBP-SCAP-Insig complex / cranial suture morphogenesis / SREBP-SCAP complex / negative regulation of steroid biosynthetic process / regulation of cholesterol biosynthetic process / sterol binding / SREBP signaling pathway / negative regulation of cholesterol biosynthetic process / COPII-coated vesicle cargo loading / regulation of fatty acid biosynthetic process / response to fatty acid / negative regulation of fatty acid biosynthetic process / Regulation of cholesterol biosynthesis by SREBP (SREBF) / oxysterol binding / response to vitamin B3 / middle ear morphogenesis / triglyceride metabolic process / inner ear morphogenesis / cholesterol biosynthetic process / roof of mouth development / cholesterol metabolic process / protein sequestering activity / ER to Golgi transport vesicle membrane / positive regulation of cholesterol biosynthetic process / response to insulin / cellular response to insulin stimulus / unfolded protein binding / response to hypoxia / immune response / Golgi membrane / endoplasmic reticulum membrane / protein-containing complex binding / endoplasmic reticulum / Golgi apparatus / membrane
Similarity search - Function
Insulin-induced protein family / Insulin-induced protein (INSIG) / : / : / SCAP N-terminal / SCAP Beta-propeller / Sterol regulatory element-binding protein cleavage-activating protein / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. ...Insulin-induced protein family / Insulin-induced protein (INSIG) / : / : / SCAP N-terminal / SCAP Beta-propeller / Sterol regulatory element-binding protein cleavage-activating protein / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
25-HYDROXYCHOLESTEROL / Sterol regulatory element-binding protein cleavage-activating protein / Insulin-induced gene 2 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsYan, R. / Cao, P. / Song, W. / Qian, H. / Du, X. / Coates, H.W. / Zhao, X. / Li, Y. / Gao, S. / Gong, X. ...Yan, R. / Cao, P. / Song, W. / Qian, H. / Du, X. / Coates, H.W. / Zhao, X. / Li, Y. / Gao, S. / Gong, X. / Liu, X. / Sui, J. / Lei, J. / Yang, H. / Brown, A.J. / Zhou, Q. / Yan, C. / Yan, N.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: Science / Year: 2021
Title: A structure of human Scap bound to Insig-2 suggests how their interaction is regulated by sterols.
Authors: Renhong Yan / Pingping Cao / Wenqi Song / Hongwu Qian / Ximing Du / Hudson W Coates / Xin Zhao / Yaning Li / Shuai Gao / Xin Gong / Ximing Liu / Jianhua Sui / Jianlin Lei / Hongyuan Yang / ...Authors: Renhong Yan / Pingping Cao / Wenqi Song / Hongwu Qian / Ximing Du / Hudson W Coates / Xin Zhao / Yaning Li / Shuai Gao / Xin Gong / Ximing Liu / Jianhua Sui / Jianlin Lei / Hongyuan Yang / Andrew J Brown / Qiang Zhou / Chuangye Yan / Nieng Yan /
Abstract: The sterol regulatory element-binding protein (SREBP) pathway controls cellular homeostasis of sterols. The key players in this pathway, Scap and Insig-1 and -2, are membrane-embedded sterol sensors. ...The sterol regulatory element-binding protein (SREBP) pathway controls cellular homeostasis of sterols. The key players in this pathway, Scap and Insig-1 and -2, are membrane-embedded sterol sensors. The 25-hydroxycholesterol (25HC)-dependent association of Scap and Insig acts as the master switch for the SREBP pathway. Here, we present cryo-electron microscopy analysis of the human Scap and Insig-2 complex in the presence of 25HC, with the transmembrane (TM) domains determined at an average resolution of 3.7 angstrom. The sterol-sensing domain in Scap and all six TMs in Insig-2 were resolved. A 25HC molecule is sandwiched between the S4 to S6 segments in Scap and TMs 3 and 4 in Insig-2 in the luminal leaflet of the membrane. Unwinding of the middle of the Scap-S4 segment is crucial for 25HC binding and Insig association.
History
DepositionMar 6, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 20, 2021Provider: repository / Type: Initial release
Revision 1.0Jan 20, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
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Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
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Revision 1.4Jun 25, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
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Assembly

Deposited unit
A: Insulin-induced gene 2 protein
B: Sterol regulatory element-binding protein cleavage-activating protein,Sterol regulatory element-binding protein cleavage-activating protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,4483
Polymers88,0452
Non-polymers4031
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3180 Å2
ΔGint-17 kcal/mol
Surface area23520 Å2

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Components

#1: Protein Insulin-induced gene 2 protein / INSIG-2


Mass: 24749.660 Da / Num. of mol.: 1 / Mutation: C14S, C90S, C215S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INSIG2 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q9Y5U4
#2: Protein Sterol regulatory element-binding protein cleavage-activating protein,Sterol regulatory element-binding protein cleavage-activating protein / SREBP cleavage-activating protein


Mass: 63295.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCAP / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q12770
#3: Chemical ChemComp-HC3 / 25-HYDROXYCHOLESTEROL / (3BETA)-CHOLEST-5-ENE-3,25-DIOL


Mass: 402.653 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Scap and Insig complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
Buffer solutionpH: 8
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_3707: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 153168 / Symmetry type: POINT
RefinementHighest resolution: 3.7 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0053222
ELECTRON MICROSCOPYf_angle_d0.7634410
ELECTRON MICROSCOPYf_dihedral_angle_d21.446478
ELECTRON MICROSCOPYf_chiral_restr0.04554
ELECTRON MICROSCOPYf_plane_restr0.006538

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