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- EMDB-22443: Cryo-EM structure of ABCG5/G8 in complex with Fab 2E10 and 11F4 -

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Basic information

Entry
Database: EMDB / ID: EMD-22443
TitleCryo-EM structure of ABCG5/G8 in complex with Fab 2E10 and 11F4
Map data
Sample
  • Complex: ABCG5/G8 in complex with Fab 2E10 and 11F4
    • Complex: ABCG5/G8
      • Protein or peptide: ATP-binding cassette sub-family G member 5
      • Protein or peptide: ATP-binding cassette sub-family G member 8
    • Complex: Fab 11F4
      • Protein or peptide: Fab 11F4 heavy chain
      • Protein or peptide: Fab 11F4 light chain
    • Complex: Fab 2E10
      • Protein or peptide: Fab 2E10 heavy chain
      • Protein or peptide: Fab 2E10 light chain
Function / homology
Function and homology information


negative regulation of intestinal phytosterol absorption / negative regulation of intestinal cholesterol absorption / Defective ABCG8 causes GBD4 and sitosterolemia / Defective ABCG5 causes sitosterolemia / sterol transport / ABC transporters in lipid homeostasis / intestinal cholesterol absorption / cholesterol transfer activity / triglyceride homeostasis / phospholipid transport ...negative regulation of intestinal phytosterol absorption / negative regulation of intestinal cholesterol absorption / Defective ABCG8 causes GBD4 and sitosterolemia / Defective ABCG5 causes sitosterolemia / sterol transport / ABC transporters in lipid homeostasis / intestinal cholesterol absorption / cholesterol transfer activity / triglyceride homeostasis / phospholipid transport / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / cholesterol efflux / response to muscle activity / bile acid signaling pathway / response to ionizing radiation / ATPase-coupled transmembrane transporter activity / ABC-type transporter activity / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / ATP-binding cassette (ABC) transporter complex / response to nutrient / cholesterol homeostasis / transmembrane transport / receptor complex / response to xenobiotic stimulus / apical plasma membrane / protein heterodimerization activity / ATP hydrolysis activity / ATP binding / metal ion binding / plasma membrane
Similarity search - Function
ABC transporter family G domain / ABC-2 type transporter / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities ...ABC transporter family G domain / ABC-2 type transporter / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-binding cassette sub-family G member 8 / ATP-binding cassette sub-family G member 5
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsHuang CS / Yu X / Min X / Wang Z / Zhang H
CitationJournal: Commun Biol / Year: 2021
Title: Cryo-EM structure of ABCG5/G8 in complex with modulating antibodies.
Authors: Hanzhi Zhang / Ching-Shin Huang / Xinchao Yu / Jonas Lee / Amit Vaish / Qing Chen / Mingyue Zhou / Zhulun Wang / Xiaoshan Min /
Abstract: The heterodimer of ATP-binding cassette transporter ABCG5 and ABCG8 mediates the excretion of sterols from liver and intestine, playing a critical role in cholesterol homeostasis. Here, we present ...The heterodimer of ATP-binding cassette transporter ABCG5 and ABCG8 mediates the excretion of sterols from liver and intestine, playing a critical role in cholesterol homeostasis. Here, we present the cryo-EM structure of ABCG5/G8 in complex with the Fab fragments from two monoclonal antibodies at 3.3Å resolution. The high-resolution structure reveals a unique dimer interface between the nucleotide-binding domains (NBD) of opposing transporters, consisting of an ordered network of salt bridges between the conserved NPXDFXXD motif and serving as a pivot point that may be important for the transport cycle. While mAb 11F4 increases the ATPase activity potentially by stabilization of the NBD dimer formation, mAb 2E10 inhibits ATP hydrolysis, likely by restricting the relative movement between the RecA and helical domain of ABCG8 NBD. Our study not only provides insights into the structural elements important for the transport cycle but also reveals novel epitopes for potential therapeutic interventions.
History
DepositionAug 11, 2020-
Header (metadata) releaseApr 7, 2021-
Map releaseApr 7, 2021-
UpdateMay 12, 2021-
Current statusMay 12, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jr7
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22443.png

Downloads & links

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Map

FileDownload / File: emd_22443.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0588 Å
Density
Contour LevelBy AUTHOR: 0.021 / Movie #1: 0.025
Minimum - Maximum-0.09167688 - 0.15695038
Average (Standard dev.)-2.3756722e-06 (±0.0033401377)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 381.168 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.05881.05881.0588
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z381.168381.168381.168
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0920.157-0.000

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Supplemental data

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Sample components

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Entire : ABCG5/G8 in complex with Fab 2E10 and 11F4

EntireName: ABCG5/G8 in complex with Fab 2E10 and 11F4
Components
  • Complex: ABCG5/G8 in complex with Fab 2E10 and 11F4
    • Complex: ABCG5/G8
      • Protein or peptide: ATP-binding cassette sub-family G member 5
      • Protein or peptide: ATP-binding cassette sub-family G member 8
    • Complex: Fab 11F4
      • Protein or peptide: Fab 11F4 heavy chain
      • Protein or peptide: Fab 11F4 light chain
    • Complex: Fab 2E10
      • Protein or peptide: Fab 2E10 heavy chain
      • Protein or peptide: Fab 2E10 light chain

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Supramolecule #1: ABCG5/G8 in complex with Fab 2E10 and 11F4

SupramoleculeName: ABCG5/G8 in complex with Fab 2E10 and 11F4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: ABCG5/G8

SupramoleculeName: ABCG5/G8 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Komagataella pastoris (fungus)

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Supramolecule #3: Fab 11F4

SupramoleculeName: Fab 11F4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #4: Fab 2E10

SupramoleculeName: Fab 2E10 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: ATP-binding cassette sub-family G member 5

MacromoleculeName: ATP-binding cassette sub-family G member 5 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 72.578406 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MGDLSSLTPG GSMGLQVNRG SQSSLEGAPA TAPEPHSLGI LHASYSVSHR VRPWWDITSC RQQWTRQILK DVSLYVESGQ IMCILGSSG SGKTTLLDAM SGRLGRAGTF LGEVYVNGRA LRREQFQDCF SYVLQSDTLL SSLTVRETLH YTALLAIRRG N PGSFQKKV ...String:
MGDLSSLTPG GSMGLQVNRG SQSSLEGAPA TAPEPHSLGI LHASYSVSHR VRPWWDITSC RQQWTRQILK DVSLYVESGQ IMCILGSSG SGKTTLLDAM SGRLGRAGTF LGEVYVNGRA LRREQFQDCF SYVLQSDTLL SSLTVRETLH YTALLAIRRG N PGSFQKKV EAVMAELSLS HVADRLIGNY SLGGISTGER RRVSIAAQLL QDPKVMLFDE PTTGLDCMTA NQIVVLLVEL AR RNRIVVL TIHQPRSELF QLFDKIAILS FGELIFCGTP AEMLDFFNDC GYPCPEHSNP FDFYMDLTSV DTQSKEREIE TSK RVQMIE SAYKKSAICH KTLKNIERMK HLKTLPMVPF KTKDSPGVFS KLGVLLRRVT RNLVRNKLAV ITRLLQNLIM GLFL LFFVL RVRSNVLKGA IQDRVGLLYQ FVGATPYTGM LNAVNLFPVL RAVSDQESQD GLYQKWQMML AYALHVLPFS VVATM IFSS VCYWTLGLHP EVARFGYFSA ALLAPHLIGE FLTLVLLGIV QNPNIVNSVV ALLSIAGVLV GSGFLRNIQE MPIPFK IIS YFTFQKYCSE ILVVNEFYGL NFTCGSSNVS VTTNPMCAFT QGIQFIEKTC PGATSRFTMN FLILYSFIPA LVILGIV VF KIRDHLISR

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Macromolecule #2: ATP-binding cassette sub-family G member 8

MacromoleculeName: ATP-binding cassette sub-family G member 8 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 75.752523 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MAGKAAEERG LPKGATPQDT SGLQDRLFSS ESDNSLYFTY SGQPNTLEVR DLNYQVDLAS QVPWFEQLAQ FKMPWTSPSC QNSCELGIQ NLSFKVRSGQ MLAIIGSSGC GRASLLDVIT GRGHGGKIKS GQIWINGQPS SPQLVRKCVA HVRQHNQLLP N LTVRETLA ...String:
MAGKAAEERG LPKGATPQDT SGLQDRLFSS ESDNSLYFTY SGQPNTLEVR DLNYQVDLAS QVPWFEQLAQ FKMPWTSPSC QNSCELGIQ NLSFKVRSGQ MLAIIGSSGC GRASLLDVIT GRGHGGKIKS GQIWINGQPS SPQLVRKCVA HVRQHNQLLP N LTVRETLA FIAQMRLPRT FSQAQRDKRV EDVIAELRLR QCADTRVGNM YVRGLSGGER RRVSIGVQLL WNPGILILDE PT SGLDSFT AHNLVKTLSR LAKGNRLVLI SLHQPRSDIF RLFDLVLLMT SGTPIYLGAA QHMVQYFTAI GYPCPRYSNP ADF YVDLTS IDRRSREQEL ATREKAQSLA ALFLEKVRDL DDFLWKAETK DLDEDTCVES SVTPLDTNCL PSPTKMPGAV QQFT TLIRR QISNDFRDLP TLLIHGAEAC LMSMTIGFLY FGHGSIQLSF MDTAALLFMI GALIPFNVIL DVISKCYSER AMLYY ELED GLYTTGPYFF AKILGELPEH CAYIIIYGMP TYWLANLRPG LQPFLLHFLL VWLVVFCCRI MALAAAALLP TFHMAS FFS NALYNSFYLA GGFMINLSSL WTVPAWISKV SFLRWCFEGL MKIQFSRRTY KMPLGNLTIA VSGDKILSVM ELDSYPL YA IYLIVIGLSG GFMVLYYVSL RFIKQKPSQD W

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Macromolecule #3: Fab 11F4 heavy chain

MacromoleculeName: Fab 11F4 heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.173945 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DVQLQESGPG LVKPSQSLSL TCTITGYSIT SDYAWNWIRQ FPGNKLEWMG YISYSGTTYY NPSLKSRFSI TRDTSRNQFF LQFNSVTTE DTGTYFCARM ITTVYYFDYW GQGTTLTVSS AKTTPPSVYP LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT W NSGSLSSG ...String:
DVQLQESGPG LVKPSQSLSL TCTITGYSIT SDYAWNWIRQ FPGNKLEWMG YISYSGTTYY NPSLKSRFSI TRDTSRNQFF LQFNSVTTE DTGTYFCARM ITTVYYFDYW GQGTTLTVSS AKTTPPSVYP LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VPSSTWPSET VTCNVAHPAS STKVDKKIVP RD

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Macromolecule #4: Fab 11F4 light chain

MacromoleculeName: Fab 11F4 light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.191529 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QIVLTQSPTI MSASLGERVT MTCTASSSAS SSYLHWYQQK PGSSPKLWIY STSNLASGVP ARFSGSGSGT SYSLTISNME AEDAATYYC HQHHRSPPTF GAGTKLELKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD ...String:
QIVLTQSPTI MSASLGERVT MTCTASSSAS SSYLHWYQQK PGSSPKLWIY STSNLASGVP ARFSGSGSGT SYSLTISNME AEDAATYYC HQHHRSPPTF GAGTKLELKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD SKDSTYSMSS TATLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRA

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Macromolecule #5: Fab 2E10 heavy chain

MacromoleculeName: Fab 2E10 heavy chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.132939 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVHLQQSGAE LVRPGTSVKM SCKAAGYTFT YWWIGWLKQR PGHGLEWIGD FFPVSGHTNF NEKFRDKATL TADTSSSTAY MQLNSLTSE DSAIYYCARK QDSSGPLYAM DYWGQGTSVT VSSAKTTPPS VYPLAPGSAA QTNSMVTLGC LVKGYFPEPV T VTWNSGSL ...String:
QVHLQQSGAE LVRPGTSVKM SCKAAGYTFT YWWIGWLKQR PGHGLEWIGD FFPVSGHTNF NEKFRDKATL TADTSSSTAY MQLNSLTSE DSAIYYCARK QDSSGPLYAM DYWGQGTSVT VSSAKTTPPS VYPLAPGSAA QTNSMVTLGC LVKGYFPEPV T VTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRD

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Macromolecule #6: Fab 2E10 light chain

MacromoleculeName: Fab 2E10 light chain / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.286709 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIQMTQSSSY LSVSLGGRVT ITCKASDHIN NWLAWYQQKP GNAPRLLISA ATSLETGVPS RFSGSGSGKD YTLSIISLQT EDVATYYCQ QYWSTPFTFG SGTKLEIKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSTTDQDS ...String:
DIQMTQSSSY LSVSLGGRVT ITCKASDHIN NWLAWYQQKP GNAPRLLISA ATSLETGVPS RFSGSGSGKD YTLSIISLQT EDVATYYCQ QYWSTPFTFG SGTKLEIKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSTTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRA

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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

BufferpH: 7.5
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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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 K2 SUMMIT (4k x 4k) / Average electron dose: 47.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 492931

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