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- EMDB-31303: Cryo-EM structure of Scap/Insig complex in the present of digitonin. -

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Basic information

Entry
Database: EMDB / ID: EMD-31303
TitleCryo-EM structure of Scap/Insig complex in the present of digitonin.
Map datacryo-EM structure of Insig-2/Scap complex in the presence of digitonin
Sample
  • Complex: Scap and Insig complex
    • Protein or peptide: Insulin-induced gene 2 protein
    • Protein or peptide: Sterol regulatory element-binding protein cleavage-activating protein
  • Ligand: Digitonin
Keywordssterol sensing / SREBP / Scap / Insig / cholestrol / digitonin / MEMBRANE PROTEIN
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 / positive regulation of cholesterol biosynthetic process / ER to Golgi transport vesicle membrane / 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
Sterol regulatory element-binding protein cleavage-activating protein / Insulin-induced gene 2 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsYan R / Cao P
Funding support China, 1 items
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, 2021-
Header (metadata) releaseJun 23, 2021-
Map releaseJun 23, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0264
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0264
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7etw
  • Surface level: 0.0264
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31303.png

Downloads & links

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Map

FileDownload / File: emd_31303.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM structure of Insig-2/Scap complex in the presence of digitonin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 200 pix.
= 218.2 Å		1.09 Å/pix.
x 200 pix.
= 218.2 Å		1.09 Å/pix.
x 200 pix.
= 218.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.091 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0264 / Movie #1: 0.0264
Minimum - Maximum-0.09787651 - 0.17102729
Average (Standard dev.)0.00021396791 (±0.0050321496)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 218.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0911.0911.091
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z218.200218.200218.200
α/β/γ90.00090.00090.000
start NX/NY/NZ192139186
NX/NY/NZ211274246
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0980.1710.000

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Supplemental data

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Sample components

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Entire : Scap and Insig complex

EntireName: Scap and Insig complex
Components
  • Complex: Scap and Insig complex
    • Protein or peptide: Insulin-induced gene 2 protein
    • Protein or peptide: Sterol regulatory element-binding protein cleavage-activating protein
  • Ligand: Digitonin

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Supramolecule #1: Scap and Insig complex

SupramoleculeName: Scap and Insig complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Insulin-induced gene 2 protein

MacromoleculeName: Insulin-induced gene 2 protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.74966 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAEGETESPG PKKSGPYISS VTSQSVNLMI RGVVLFFIGV FLALVLNLLQ IQRNVTLFPP DVIASIFSSA WWVPPCCGTA SAVIGLLYP SIDRHLGEPH KFKREWSSVM RCVAVFVGIN HASAKVDFDN NIQLSLTLAA LSIGLWWTFD RSRSGFGLGV G IAFLATVV ...String:
MAEGETESPG PKKSGPYISS VTSQSVNLMI RGVVLFFIGV FLALVLNLLQ IQRNVTLFPP DVIASIFSSA WWVPPCCGTA SAVIGLLYP SIDRHLGEPH KFKREWSSVM RCVAVFVGIN HASAKVDFDN NIQLSLTLAA LSIGLWWTFD RSRSGFGLGV G IAFLATVV TQLLVYNGVY QYTSPDFLYV RSWLPCIFFA GGITMGNIGR QLAMYESKVI AEKSHQE

UniProtKB: Insulin-induced gene 2 protein

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Macromolecule #2: Sterol regulatory element-binding protein cleavage-activating protein

MacromoleculeName: Sterol regulatory element-binding protein cleavage-activating protein
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 81.863531 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTLTERLREK ISRAFYNHGL LCASYPIPII LFTGFCILAC CYPLLKLPLP GTGPVEFTTP VKDYSPPPVD SDRKQGEPTE QPEWYVGAP VAYVQQIFVK SSVFPWHKNL LAVDVFRSPL SRAFQLVEEI RNHVLRDSSG IRSLEELCLQ VTDLLPGLRK L RNLLPEHG ...String:
MTLTERLREK ISRAFYNHGL LCASYPIPII LFTGFCILAC CYPLLKLPLP GTGPVEFTTP VKDYSPPPVD SDRKQGEPTE QPEWYVGAP VAYVQQIFVK SSVFPWHKNL LAVDVFRSPL SRAFQLVEEI RNHVLRDSSG IRSLEELCLQ VTDLLPGLRK L RNLLPEHG CLLLSPGNFW QNDWERFHAD PDIIGTIHQH EPKTLQTSAT LKDLLFGVPG KYSGVSLYTR KRMVSYTITL VF QHYHAKF LGSLRARLML LHPSPNCSLR AESLVHVHFK EEIGVAELIP LVTTYIILFA YIYFSTRKID MVKSKWGLAL AAV VTVLSS LLMSVGLCTL FGLTPTLNGG EIFPYLVVVI GLENVLVLTK SVVSTPVDLE VKLRIAQGLS SESWSIMKNM ATEL GIILI GYFTLVPAIQ EFCLFAVVGL VSDFFLQMLF FTTVLSIDIR RMELADLNKR LPPEACLPSA KPVGQPTRYE RQLAV RPST PHTITLQPSS FRNLRLPKRL RVVYFLARTR LAQRLIMAGT VVWIGILVYT DPAGLRNYLA AQVTEQSPLG EGALAP MPV PSGMLPPSHP DPAFSIFPPD APKLPENQTS PGESPERGGP AEVVHDSPVP EVTWGPEDEE LWRKLSFRHW PTLFSYY NI TLAKRYISLL PVIPVTLRLN PREALEGRHP QDGRSAWPPP GPIPAGHWEA GPKGPGGVQA HGDVTLYKVA ALGLATGI V LVLLLLCLYR VLCP

UniProtKB: Sterol regulatory element-binding protein cleavage-activating protein

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Macromolecule #4: Digitonin

MacromoleculeName: Digitonin / type: ligand / ID: 4 / Number of copies: 4 / Formula: AJP
Molecular weightTheoretical: 1.229312 KDa
Chemical component information

ChemComp-AJP:
Digitonin / detergent*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 252929
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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