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- PDB-7etw: Cryo-EM structure of Scap/Insig complex in the present of digitonin. -

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Basic information

Entry
Database: PDB / ID: 7etw
TitleCryo-EM structure of Scap/Insig complex in the present of digitonin.
Components
  • Insulin-induced gene 2 protein
  • Sterol regulatory element-binding protein cleavage-activating protein
KeywordsMEMBRANE PROTEIN / sterol sensing / SREBP / Scap / Insig / cholestrol / digitonin
Function / homology
Function and homology information


SREBP-SCAP complex retention in endoplasmic reticulum / SREBP-SCAP-Insig complex / cranial suture morphogenesis / SREBP-SCAP complex / regulation of cholesterol biosynthetic process / negative regulation of steroid biosynthetic process / cellular lipid metabolic process / response to vitamin B3 / sterol binding / SREBP signaling pathway ...SREBP-SCAP complex retention in endoplasmic reticulum / SREBP-SCAP-Insig complex / cranial suture morphogenesis / SREBP-SCAP complex / regulation of cholesterol biosynthetic process / negative regulation of steroid biosynthetic process / cellular lipid metabolic process / response to vitamin B3 / sterol binding / SREBP signaling pathway / COPII-coated vesicle cargo loading / regulation of fatty acid biosynthetic process / negative regulation of fatty acid biosynthetic process / positive regulation of cholesterol biosynthetic process / Regulation of cholesterol biosynthesis by SREBP (SREBF) / oxysterol binding / negative regulation of cholesterol biosynthetic process / middle ear morphogenesis / inner ear morphogenesis / triglyceride metabolic process / roof of mouth development / cholesterol biosynthetic process / protein sequestering activity / cholesterol metabolic process / response to insulin / ER to Golgi transport vesicle membrane / cellular response to insulin stimulus / unfolded protein binding / response to hypoxia / immune response / Golgi membrane / protein-containing complex binding / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / membrane
Similarity search - Function
Insulin-induced protein family / Insulin-induced protein (INSIG) / 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. ...Insulin-induced protein family / Insulin-induced protein (INSIG) / 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. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
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: 4.1 Å
AuthorsYan, R. / Cao, P. / Song, W. / Li, Y. / Wang, T. / Qian, H. / Yan, C. / Yan, N.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: Cell Rep / Year: 2021
Title: Structural basis for sterol sensing by Scap and Insig.
Authors: Renhong Yan / Pingping Cao / Wenqi Song / Yaning Li / Tongtong Wang / Hongwu Qian / Chuangye Yan / Nieng Yan /
Abstract: The sterol regulatory element-binding protein (SREBP) pathway monitors the cellular cholesterol level through sterol-regulated association between the SREBP cleavage-activating protein (Scap) and the ...The sterol regulatory element-binding protein (SREBP) pathway monitors the cellular cholesterol level through sterol-regulated association between the SREBP cleavage-activating protein (Scap) and the insulin-induced gene (Insig). Despite structural determination of the Scap and Insig-2 complex bound to 25-hydroxycholesterol, the luminal domains of Scap remain unresolved. In this study, combining cryogenic electron microscopy (cryo-EM) analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex purified in digitonin. The luminal domain loop 1 and a co-folded segment in loop 7 of Scap resemble those of the luminal/extracellular domain in NPC1 and related proteins, providing clues to the cholesterol-regulated interaction of loop 1 and loop 7. An additional luminal interface is observed between Scap and Insig. We also show that Scap(D428A), which inhibits SREBP activation even under sterol depletion, exhibits an identical conformation with the wild-type protein when complexed with Insig-2, and its constitutive suppression of the SREBP pathway may also involve a later step in protein trafficking.
History
DepositionMay 14, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 23, 2021Provider: repository / Type: Initial release
Revision 2.0Sep 22, 2021Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / database_2 / database_PDB_caveat / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_validate_chiral
Item: _atom_site.auth_comp_id / _atom_site.label_comp_id ..._atom_site.auth_comp_id / _atom_site.label_comp_id / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_entity_instance_feature.auth_comp_id / _pdbx_entity_instance_feature.comp_id / _pdbx_entity_nonpoly.comp_id / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id
Revision 2.1Mar 2, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.year

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)111,9557
Polymers106,6132
Non-polymers5,3425
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3280 Å2
ΔGint-22 kcal/mol
Surface area42880 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 / SCAP / SREBP cleavage-activating protein


Mass: 81863.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCAP, KIAA0199, PSEC0227 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q12770
#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Chemical
ChemComp-AJP / Digitonin / Digitonin


Mass: 1229.312 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C56H92O29 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
Has ligand of interestY

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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: HEK293S
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 FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
4GctfCTF correction
13RELION33D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 252929 / Symmetry type: POINT

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