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- EMDB-23407: Map of full mutant cScap/Fab complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23407
TitleMap of full mutant cScap/Fab complex
Map dataMap of Scap(D435VB)/Insig-1/4G10 complex complex. Low pass filtered by local resolution
Sample
  • Complex: Ternary complex of Scap with Fab fragment
    • Complex: Scap
      • Protein or peptide: Sterol regulatory element-binding protein cleavage-activating protein
      • Protein or peptide: Insig-1
    • Complex: 4G10 Fab
      • Protein or peptide: 4G10 Fab light chain
      • Protein or peptide: 4G10 Fab heavy chain
Function / homology
Function and homology information


Regulation of cholesterol biosynthesis by SREBP (SREBF) / SREBP-SCAP complex / regulation of cholesterol biosynthetic process / SREBP signaling pathway / cellular lipid metabolic process / sterol binding / 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 / SREBP signaling pathway / cellular lipid metabolic process / sterol binding / regulation of fatty acid biosynthetic process / positive regulation of cholesterol biosynthetic process / cholesterol metabolic process / response to insulin / ER to Golgi transport vesicle membrane / membrane => GO:0016020 / 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. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat ...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. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / 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) / Mouse (mice)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsKober DL / Radhakrishnan A / Goldstein JL / Brown MS / Clark LD / Bai X-C / Rosenbaum DM
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 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 Scholar Award United States
Leducq Foundation19CVD04 France
Cancer Prevention and Research Institute of Texas (CPRIT)RR160082 United States
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-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0369
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0369
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23407.png

Downloads & links

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Map

FileDownload / File: emd_23407.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of Scap(D435VB)/Insig-1/4G10 complex complex. Low pass filtered by local resolution
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.0369 / Movie #1: 0.0369
Minimum - Maximum-0.044015773 - 0.12214054
Average (Standard dev.)3.339913e-05 (±0.003398625)
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/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS270270270
D min/max/mean-0.0440.1220.000

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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
      • Protein or peptide: Sterol regulatory element-binding protein cleavage-activating protein
      • Protein or peptide: Insig-1
    • Complex: 4G10 Fab
      • Protein or peptide: 4G10 Fab light chain
      • Protein or peptide: 4G10 Fab heavy 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

SupramoleculeName: Scap / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: 4G10 Fab

SupramoleculeName: 4G10 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
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 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: PAMTLTEKLR ERISRAFYNH GLLCASYPIP IILFTGLCIL ACCYPLLKLP LPGTGPVEFS TPVKDYFPPS PDVVSQQGDL SERPDWYVGA PVAYIQQIFV KATVSPWQKN FLAVDVFRSP LSRVFQLVEE IRNHALRDSS GVKSLEEVCL QVTDLLPGLK KLRNLLPEHG ...String:
PAMTLTEKLR ERISRAFYNH GLLCASYPIP IILFTGLCIL ACCYPLLKLP LPGTGPVEFS TPVKDYFPPS PDVVSQQGDL SERPDWYVGA PVAYIQQIFV KATVSPWQKN FLAVDVFRSP LSRVFQLVEE IRNHALRDSS GVKSLEEVCL QVTDLLPGLK KLRNLLPEHG CLLLSPGNFW QNDRERFNAD PDIIKTIHQH EPKALQTSAT LKDLLFGLPG KYSGVNLYNR KRVVSYTVTL GLQRYDSRFL SSLRSRLKLL HPSPNCTLRE DSIVHVHFKE EIGIAELIPL VTTYIILFAY IYFSTRKIDM VKSKWGLALA AVVTVLSSLL MSVGLCTLFG LTPTLNGGSV PVSFREIFPY LVVVIGLENV LVLTKSVVST PVDLEVKLRI AQGLSNESWS IMKNMATELG IILIGYFTLV PAIQEFCLFA VVGLVSVFFL QMLFFTTVLS IDIRRMELAD LNKRLPAEAC LPPAKPASRS QRYERQPAVR PATPHTITLQ PSSFRNLRLP KRLRVIYFFA RTRLAQRLIM AGTVIWIGIL VYTDPAGLRT YLTSQVTEQS PLGEAGLPPM PVPGGVLPAG DPKIDLSVFP SDPIQLSENQ TQQREQQAGL EPLGRLETNQ HSWAQGPEGR GNGQTELGTE AEVTWGAEDE EIWRKLSFRH WPSLFSYYNI TLAKRYISIL PAIPVTLYLN PQEALEVRHP QEANRYHPFL SSSGGKLNAE AQPDQTSSRL QGHRDVTLYK VAALGLASGI LLVLLLFCLY RLLCPKNYGQ NGLSHSRRRR GDLPCDDYGY SPPETEIVPL VLRGHLMDIE CLASDGMLLV SCCLVGQIRV WDAQTGDCLT VIPKPRLRRD SSGIFDYQES WDHSPDGKTG LDDSFESSHQ LKRMLSPPQP PLFCDQPDLT SLIDTNFSEQ VKVAESEPRL RAVGGRQKEA GYDFSSLVGK VYEEHSTSNC MNFGGLSAPH GQAGFCVGGS TARSLGCGSE EGGCGGRRRS LGDESLSGFD KSSPLPSWGG DFESSVWSLD LQGNLIVAGR SNGKLEVWDA IEGTLRSSND ESQSGITALV FLNNRIVAAR LNGSLDFFSL ETHTSLNHLQ FRGAPSRSSI PSSPLFSSSD VIVCQLTHTV SCAHQKPITA LKAAAGRLVT GSQDHTLRVF RLEDSCCLFT LQGHSGAITA VYIDQTMVLA SGGQDGAICL WDVLTGSKVS HMYAHRGDVT SLTCTTSCVI SSGLDDVISI WDRSSGIKLY SIQQEMGCGS SLGVISDNLL VTGGQGCVSF WDIGYGDLLQ TVYLGKSNES QPARQILVLE NAAIVCNFGS ELSLVYVPSV LEKLDDYKDD DDKGSDYKDD DDKGSDYKDD DDK

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Macromolecule #2: Insig-1

MacromoleculeName: Insig-1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTGPKADAMQ SPSPSAGRAE REASGGSATT WRQHLVQRSV VLFVVGAFMA LVLNLLQIQR NVTLFPDEVI ATLFSSAWWV PPCCGTAAAV VGLLYPCIDS HLGEPHKFKR EWASVMRCIA VFVGINHASA KLDFANNVQL SLTLAALSLG LWWTFDRSRS GLGLGITIAF ...String:
MTGPKADAMQ SPSPSAGRAE REASGGSATT WRQHLVQRSV VLFVVGAFMA LVLNLLQIQR NVTLFPDEVI ATLFSSAWWV PPCCGTAAAV VGLLYPCIDS HLGEPHKFKR EWASVMRCIA VFVGINHASA KLDFANNVQL SLTLAALSLG LWWTFDRSRS GLGLGITIAF VATLITQFLV YNGVYQYTSP DFLYIRSWLP CIFFSGGVTV GNIGRQLAMG IPEKPHNDSR ENLYFQGGSG ATNFSLLKQA GDVEENPG

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Macromolecule #3: 4G10 Fab light chain

MacromoleculeName: 4G10 Fab light chain / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mouse (mice)
SequenceString: DIQMTQTTSS LSASLGDRVT ISCRASQDIR NYLNWYQQKP DGTVKLLIYY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQQ TNTLPWTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD ...String:
DIQMTQTTSS LSASLGDRVT ISCRASQDIR NYLNWYQQKP DGTVKLLIYY TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQQ TNTLPWTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

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Macromolecule #4: 4G10 Fab heavy chain

MacromoleculeName: 4G10 Fab heavy chain / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Mouse (mice)
SequenceString: TGVHSEVQLQ QSGAELVRPG ASVKLSCTAS GFKIKDDYIH WVKQRPEQGL EWIGRIDPAN GHTRYAPKFQ DKATITADTS SNTAYLQLSS LTSEDTAVYY CTRYNDYDAF YFDYWGQGTT LTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA ...String:
TGVHSEVQLQ QSGAELVRPG ASVKLSCTAS GFKIKDDYIH WVKQRPEQGL EWIGRIDPAN GHTRYAPKFQ DKATITADTS SNTAYLQLSS LTSEDTAVYY CTRYNDYDAF YFDYWGQGTT LTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KRVEPKSCDK T

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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 chloridesodium 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 9052025
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 80678
FSC plot (resolution estimation)

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