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- PDB-7lkh: Chicken Scap D435V L1-L7 domain / Fab complex focused map -

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Basic information

Entry
Database: PDB / ID: 7lkh
TitleChicken Scap D435V L1-L7 domain / Fab complex focused map
Components
  • 4G10 Fab heavy chain
  • 4G10 Fab kappa chain
  • Sterol regulatory element-binding protein cleavage-activating protein
KeywordsLIPID BINDING PROTEIN / Cholesterol
Function / homology
Function and homology information


Regulation of cholesterol biosynthesis by SREBP (SREBF) / SREBP-SCAP complex / regulation of cholesterol biosynthetic process / : / sterol binding / SREBP signaling pathway / regulation of fatty acid biosynthetic process / positive regulation of cholesterol biosynthetic process / cholesterol metabolic process / response to insulin ...Regulation of cholesterol biosynthesis by SREBP (SREBF) / SREBP-SCAP complex / regulation of cholesterol biosynthetic process / : / sterol binding / SREBP signaling pathway / regulation of fatty acid biosynthetic process / positive regulation of cholesterol biosynthetic process / cholesterol metabolic process / response to insulin / ER to Golgi transport vesicle membrane / response to hypoxia / immune response / Golgi membrane / endoplasmic reticulum membrane
Similarity search - Function
Sterol regulatory element-binding protein cleavage-activating protein / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / Quinoprotein alcohol dehydrogenase-like superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. ...Sterol regulatory element-binding protein cleavage-activating protein / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / Quinoprotein alcohol dehydrogenase-like superfamily / 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/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Sterol regulatory element-binding protein cleavage-activating protein
Similarity search - Component
Biological speciesGallus gallus (chicken)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKober, D.L. / Radhakrishnan, A. / Goldstein, J.L. / Brown, M.S. / Clark, L.D. / Bai, X.-C. / Rosenbaum, D.M.
Funding support United States, France, 11items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM116387 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01HL020948 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM136976 United States
Welch FoundationI-1770 United States
Welch FoundationI-1793 United States
Welch FoundationI-1944 United States
Mallinckrodt Foundation United States
Leducq Foundation19CVD04 France
Cancer Prevention and Research Institute of Texas (CPRIT)RR160082 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP170644 United States
American Heart Association18POST34080141 United States
CitationJournal: Cell / Year: 2021
Title: Scap structures highlight key role for rotation of intertwined luminal loops in cholesterol sensing.
Authors: Daniel L Kober / Arun Radhakrishnan / Joseph L Goldstein / Michael S Brown / Lindsay D Clark / Xiao-Chen Bai / Daniel M Rosenbaum /
Abstract: The cholesterol-sensing protein Scap induces cholesterol synthesis by transporting membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) from the endoplasmic ...The cholesterol-sensing protein Scap induces cholesterol synthesis by transporting membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi apparatus for proteolytic activation. Transport requires interaction between Scap's two ER luminal loops (L1 and L7), which flank an intramembrane sterol-sensing domain (SSD). Cholesterol inhibits Scap transport by binding to L1, which triggers Scap's binding to Insig, an ER retention protein. Here we used cryoelectron microscopy (cryo-EM) to elucidate two structures of full-length chicken Scap: (1) a wild-type free of Insigs and (2) mutant Scap bound to chicken Insig without cholesterol. Strikingly, L1 and L7 intertwine tightly to form a globular domain that acts as a luminal platform connecting the SSD to the rest of Scap. In the presence of Insig, this platform undergoes a large rotation accompanied by rearrangement of Scap's transmembrane helices. We postulate that this conformational change halts Scap transport of SREBPs and inhibits cholesterol synthesis.
History
DepositionFeb 2, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 30, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Sterol regulatory element-binding protein cleavage-activating protein
H: 4G10 Fab heavy chain
L: 4G10 Fab kappa chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,3994
Polymers196,9753
Non-polymers4241
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3660 Å2
ΔGint-5 kcal/mol
Surface area24710 Å2

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Components

#1: Protein Sterol regulatory element-binding protein cleavage-activating protein


Mass: 148364.828 Da / Num. of mol.: 1 / Mutation: D435V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: SCAP / Production host: Homo sapiens (human) / References: UniProt: A0A3Q3ANV4
#2: Antibody 4G10 Fab heavy chain


Mass: 24942.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Plasmid details: Hybridoma line
#3: Antibody 4G10 Fab kappa chain


Mass: 23667.178 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Plasmid details: Hybridoma cell line
#4: 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
Has ligand of interestN
Has protein modificationY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Ternary complex of Scap with Fab fragmentCOMPLEXFab fragment generated by proteolytic cleavage of IgG antibody#1-#30MULTIPLE SOURCES
2Scap L1-L7 domainCOMPLEX#11RECOMBINANT
34G10 FabCOMPLEXFab fragment generated by proteolytic cleavage of IgG antibody#2-#31NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11NO
210.172 MDaNO
310.05 MDaNO
13
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Gallus gallus (chicken)9031
23Mus musculus (house mouse)10090
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMHEPES1
30.004 %glyco-diosgenin1
40.0004 %cholestoryl hemisuccinate1
50.5 mMTris(2-carboxyethyl)phosphine hydrochloride1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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: 1.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9052025
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 155187 / Symmetry type: POINT

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