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- EMDB-9940: Cryo-EM structure of the human P4-type flippase ATP8A1-CDC50 (E2P... -

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Basic information

Entry
Database: EMDB / ID: EMD-9940
TitleCryo-EM structure of the human P4-type flippase ATP8A1-CDC50 (E2P state class3)
Map data
Sample
  • Complex: ATP8A1-CDC50a
    • Protein or peptide: ATP8A1
    • Protein or peptide: CDC50a
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsHiraizumi M / Yamashita K / Nishizawa T / Nureki O
CitationJournal: Science / Year: 2019
Title: Cryo-EM structures capture the transport cycle of the P4-ATPase flippase.
Authors: Masahiro Hiraizumi / Keitaro Yamashita / Tomohiro Nishizawa / Osamu Nureki /
Abstract: In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is ...In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is critical for membrane trafficking and signaling pathways. Here, we report the cryo-electron microscopy structures of six distinct intermediates of the human ATP8A1-CDC50a heterocomplex at resolutions of 2.6 to 3.3 angstroms, elucidating the lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.
History
DepositionJun 7, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseAug 28, 2019-
UpdateFeb 10, 2021-
Current statusFeb 10, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9940.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.19066048 - 0.24813984
Average (Standard dev.)0.00022503239 (±0.0059419395)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 215.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z215.800215.800215.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.1910.2480.000

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Supplemental data

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Mask #1

Fileemd_9940_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_9940_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_9940_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : ATP8A1-CDC50a

EntireName: ATP8A1-CDC50a
Components
  • Complex: ATP8A1-CDC50a
    • Protein or peptide: ATP8A1
    • Protein or peptide: CDC50a

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Supramolecule #1: ATP8A1-CDC50a

SupramoleculeName: ATP8A1-CDC50a / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant plasmid: pcDNA3.4

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Macromolecule #1: ATP8A1

MacromoleculeName: ATP8A1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSREFMPTMR RTVSEIRSRA EGYEKTDDVS EKTSLADQEE VRTIFINQPQ LTKFCNNHVS TAKYNIITF LPRFLYSQFR RAANSFFLFI ALLQQIPDVS PTGRYTTLVP LLFILAVAAI K EIIEDIKR HKADNAVNKK QTQVLRNGAW EIVHWEKVNV GDIVIIKGKE ...String:
GSREFMPTMR RTVSEIRSRA EGYEKTDDVS EKTSLADQEE VRTIFINQPQ LTKFCNNHVS TAKYNIITF LPRFLYSQFR RAANSFFLFI ALLQQIPDVS PTGRYTTLVP LLFILAVAAI K EIIEDIKR HKADNAVNKK QTQVLRNGAW EIVHWEKVNV GDIVIIKGKE YIPADTVLLS SS EPQAMCY IETSNLDGET NLKIRQGLPA TSDIKDVDSL MRISGRIECE SPNRHLYDFV GNI RLDGHG TVPLGADQIL LRGAQLRNTQ WVHGIVVYTG HDTKLMQNST SPPLKLSNVE RITN VQILI LFCILIAMSL VCSVGSAIWN RRHSGKDWYL NLNYGGASNF GLNFLTFIIL FNNLI PISL LVTLEVVKFT QAYFINWDLD MHYEPTDTAA MARTSNLNEE LGQVKYIFSD KTGTLT CNV MQFKKCTIAG VAYGQNSQFG DEKTFSDSSL LENLQNNHPT APIICEFLTM MAVCHTA VP ERERDKIIYQ AASPDEGALV RAAKQLNFVF TGRTPDSVII DSLGQEERYE LLNVLEFT S ARKRMSVIVR TPSGKLRLYC KGADTVIYDR LAETSKYKEI TLKHLEQFAT EGLRTLCFA VAEISESDFQ EWRAVYQRAS TSVQNRLLKL EESYELIEKN LQLLGATAIE DKLQDQVPET IETLMKADI KIWILTGDKQ ETAINIGHSC KLLKKNMGMI VINEGSLDGT RETLSRHCTT L GDALRKEN DFALIIDGKT LKYALTFGVR QYFLDLALSC KAVICCRVSP LQKSEVVEMV KK QVKVVTL AIGDGANDVS MIQTAHVGVG ISGNEGLQAA NSSDYSIAQF KYLKNLLMIH GAW NYNRVS KCILYCFYKN IVLYIIEIWF AFVNGFSGQI LFERWCIGLY NVMFTAMPPL TLGI FERSC RKENMLKYPE LYKTSQNALD FNTKVFWVHC LNGLFHSVIL FWFPLKALQY GTAFG NGKT SDYLLLGNFV YTFVVITVCL KAGLETSYWT WFSHIAIWGS IALWVVFFGI YSSLWP AIP MAPDMSGEAA MLFSSGVFWM GLLFIPVASL LLDVVYKVIK RTAFKTLVDE VQELEAK SQ DPGAVVLGKS LTERAQLLKN VFKKNHVNLY RSESLQQNLL HGYAFSQDEN GIVSQSEV I RAYDTTKQRP DEW

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Macromolecule #2: CDC50a

MacromoleculeName: CDC50a / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAMNYNAKDE VDGGPPCAPG GTAKTRRPDN TAFKQQRLPA WQPILTAGTV LPIFFIIGLI FIPIGIGIF VTSNNIREIE IDYTGTEPSS PCNKCLSPDV TPCFCTINFT LEKSFEGNVF M YYGLSNFY QNHRRYVKSR DDSQLNGDSS ALLNPSKECE PYRRNEDKPI ...String:
MAMNYNAKDE VDGGPPCAPG GTAKTRRPDN TAFKQQRLPA WQPILTAGTV LPIFFIIGLI FIPIGIGIF VTSNNIREIE IDYTGTEPSS PCNKCLSPDV TPCFCTINFT LEKSFEGNVF M YYGLSNFY QNHRRYVKSR DDSQLNGDSS ALLNPSKECE PYRRNEDKPI APCGAIANSM FN DTLELFL IGNDSYPIPI ALKKKGIAWW TDKNVKFRNP PGGDNLEERF KGTTKPVNWL KPV YMLDSD PDNNGFINED FIVWMRTAAL PTFRKLYRLI ERKSDLHPTL PAGRYSLNVT YNYP VHYFD GRKRMILSTI SWMGGKNPFL GIAYIAVGSI SFLLGVVLLV INHKYRNSSN TADIT I

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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

Concentration4 mg/mL
BufferpH: 7.5
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: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 64.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 481661
FSC plot (resolution estimation)

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