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- EMDB-23405: WT Chicken Scap L1-L7 / Fab 4G10 complex focused refinement -

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Basic information

Entry
Database: EMDB / ID: EMD-23405
TitleWT Chicken Scap L1-L7 / Fab 4G10 complex focused refinement
Map dataFocused classification map of WT Scap L1-L7/Fab
Sample
  • Complex: Ternary complex of Scap with Fab fragment
    • Complex: Scap L1-L7 domain
      • Protein or peptide: Sterol regulatory element-binding protein cleavage-activating protein
    • Complex: 4G10 Fab, 4G10 light chain
      • Protein or peptide: 4G10 heavy chain
      • Protein or peptide: 4G10 light chain
KeywordsCholesterol / LIPID BINDING PROTEIN / SREBP
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsKober DL / Radhakrishnan A
Funding support United States, France, 11 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM116387 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM136976 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01HL020948 United States
Welch FoundationI-1770 United States
Welch FoundationI-1793 United States
Welch FoundationI-1944 United States
Mallinckrodt Foundation United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR160082 United States
Leducq Foundation19CVD04 France
American Heart Association18POST34080141 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP170644 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, 2021-
Header (metadata) releaseJun 30, 2021-
Map releaseJun 30, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0537
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0537
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7lkf
  • Surface level: 0.0537
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23405.png

Downloads & links

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Map

FileDownload / File: emd_23405.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused classification map of WT Scap L1-L7/Fab
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 270 pix.
= 291.6 Å		1.08 Å/pix.
x 270 pix.
= 291.6 Å		1.08 Å/pix.
x 270 pix.
= 291.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0537 / Movie #1: 0.0537
Minimum - Maximum-0.20536686 - 0.30715764
Average (Standard dev.)-0.000053366453 (±0.0048753116)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions270270270
Spacing270270270
CellA=B=C: 291.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z270270270
origin x/y/z0.0000.0000.000
length x/y/z291.600291.600291.600
α/β/γ90.00090.00090.000
start NX/NY/NZ192139186
NX/NY/NZ211274246
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS270270270
D min/max/mean-0.2050.307-0.000

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Supplemental data

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Sample components

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Entire : Ternary complex of Scap with Fab fragment

EntireName: Ternary complex of Scap with Fab fragment
Components
  • Complex: Ternary complex of Scap with Fab fragment
    • Complex: Scap L1-L7 domain
      • Protein or peptide: Sterol regulatory element-binding protein cleavage-activating protein
    • Complex: 4G10 Fab, 4G10 light chain
      • Protein or peptide: 4G10 heavy chain
      • Protein or peptide: 4G10 light chain

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Supramolecule #1: Ternary complex of Scap with Fab fragment

SupramoleculeName: Ternary complex of Scap with Fab fragment / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fab fragment generated by proteolytic cleavage of IgG antibody
Molecular weightTheoretical: 50 KDa

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Supramolecule #2: Scap L1-L7 domain

SupramoleculeName: Scap L1-L7 domain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)

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Supramolecule #3: 4G10 Fab, 4G10 light chain

SupramoleculeName: 4G10 Fab, 4G10 light chain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: Fab fragment generated by proteolytic cleavage of IgG antibody
Source (natural)Organism: Mus musculus (house mouse)

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

MacromoleculeName: Sterol regulatory element-binding protein cleavage-activating protein
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 148.212594 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTLTEKLRER ISRAFYNHGL LCASYPIPII LFTGLCILAC CYPLLKLPLP GTGPVEFSTP VKDYFPPSPD VVSQQGDLSE RPDWYVGAP VAYIQQIFVK ATVSPWQKNF LAVDVFRSPL SRVFQLVEEI RNHALRDSSG VKSLEEVCLQ VTDLLPGLKK L RNLLPEHG ...String:
MTLTEKLRER ISRAFYNHGL LCASYPIPII LFTGLCILAC CYPLLKLPLP GTGPVEFSTP VKDYFPPSPD VVSQQGDLSE RPDWYVGAP VAYIQQIFVK ATVSPWQKNF LAVDVFRSPL SRVFQLVEEI RNHALRDSSG VKSLEEVCLQ VTDLLPGLKK L RNLLPEHG CLLLSPGNFW QNDRERFNAD PDIIKTIHQH EPKALQTSAT LKDLLFGLPG KYSGVNLYNR KRVVSYTVTL GL QRYDSRF LSSLRSRLKL LHPSPNCTLR EDSIVHVHFK EEIGIAELIP LVTTYIILFA YIYFSTRKID MVKSKWGLAL AAV VTVLSS LLMSVGLCTL FGLTPTLNGG EIFPYLVVVI GLENVLVLTK SVVSTPVDLE VKLRIAQGLS NESWSIMKNM ATEL GIILI GYFTLVPAIQ EFCLFAVVGL VSDFFLQMLF FTTVLSIDIR RMELADLNKR LPAEACLPPA KPASRSQRYE RQPAV RPAT PHTITLQPSS FRNLRLPKRL RVIYFFARTR LAQRLIMAGT VIWIGILVYT DPAGLRTYLT SQVTEQSPLG EAGLPP MPV PGGVLPAGDP KIDLSVFPSD PIQLSENQTQ QREQQAGLEP LGRLETNQHS WAQGPEGRGN GQTELGTEAE VTWGAED EE IWRKLSFRHW PSLFSYYNIT LAKRYISILP AIPVTLYLNP QEALEVRHPQ EANRYHPFLS SSGGKLNAEA QPDQTSSR L QGHRDVTLYK VAALGLASGI LLVLLLFCLY RLLCPKNYGQ NGLSHSRRRR GDLPCDDYGY SPPETEIVPL VLRGHLMDI ECLASDGMLL VSCCLVGQIR VWDAQTGDCL TVIPKPRLRR DSSGIFDYQE SWDHSPDGKT GLDDSFESSH QLKRMLSPPQ PPLFCDQPD LTSLIDTNFS EQVKVAESEP RLRAVGGRQK EAGYDFSSLV GKVYEEHSTS NCMNFGGLSA PHGQAGFCVG G STARSLGC GSEEGGCGGR RRSLGDESLS GFDKSSPLPS WGGDFESSVW SLDLQGNLIV AGRSNGKLEV WDAIEGTLRS SN DESQSGI TALVFLNNRI VAARLNGSLD FFSLETHTSL NHLQFRGAPS RSSIPSSPLF SSSDVIVCQL THTVSCAHQK PIT ALKAAA GRLVTGSQDH TLRVFRLEDS CCLFTLQGHS GAITAVYIDQ TMVLASGGQD GAICLWDVLT GSKVSHMYAH RGDV TSLTC TTSCVISSGL DDVISIWDRS SGIKLYSIQQ EMGCGSSLGV ISDNLLVTGG QGCVSFWDIG YGDLLQTVYL GKSNE SQPA RQILVLENAA IVCNFGSELS LVYVPSVLEK LDDYKDDDDK GSDYKDDDDK GSDYKDDDDK

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

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Macromolecule #2: 4G10 heavy chain

MacromoleculeName: 4G10 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.94266 KDa
SequenceString: TGVHSEVQLQ QSGAELVRPG ASVKLSCTAS GFKIKDDYIH WVKQRPEQGL EWIGRIDPAN GHTRYAPKFQ DKATITADTS SNTAYLQLS SLTSEDTAVY YCTRYNDYDA FYFDYWGQGT TLTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP E PVTVSWNS ...String:
TGVHSEVQLQ QSGAELVRPG ASVKLSCTAS GFKIKDDYIH WVKQRPEQGL EWIGRIDPAN GHTRYAPKFQ DKATITADTS SNTAYLQLS SLTSEDTAVY YCTRYNDYDA FYFDYWGQGT TLTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP E PVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKT

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Macromolecule #3: 4G10 light chain

MacromoleculeName: 4G10 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.667178 KDa
SequenceString: DIQMTQTTSS LSASLGDRVT ISCRASQDIR NYLNWYQQKP DGTVKLLIYY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQ QTNTLPWTFG GGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIQMTQTTSS LSASLGDRVT ISCRASQDIR NYLNWYQQKP DGTVKLLIYY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQ QTNTLPWTFG GGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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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

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPES
0.004 %glyco-diosgenin
0.0004 %cholestoryl hemisuccinate
0.5 mMTris(2-carboxyethyl)phosphine hydrochloride
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.6 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

Particle selectionNumber selected: 9052025
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 223673
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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