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- EMDB-9933: Cryo-EM structure of the human P4-type flippase ATP8A1-CDC50 (E1 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9933
TitleCryo-EM structure of the human P4-type flippase ATP8A1-CDC50 (E1 state class3)
Map data
SampleATP8A1-CDC50a:
ATP8A1 / CDC50a
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 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-
UpdateNov 13, 2019-
Current statusNov 13, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_9933.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 260 pix.
= 215.8 Å
0.83 Å/pix.
x 260 pix.
= 215.8 Å
0.83 Å/pix.
x 260 pix.
= 215.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.080491826 - 0.14312711
Average (Standard dev.)0.00024334615 (±0.0036349634)
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
start NX/NY/NZ000
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.0800.1430.000

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Supplemental data

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Sample components

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Entire ATP8A1-CDC50a

EntireName: ATP8A1-CDC50a / Number of components: 3

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Component #1: protein, ATP8A1-CDC50a

ProteinName: ATP8A1-CDC50a / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Vector: pcDNA3.4

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Component #2: protein, ATP8A1

ProteinName: ATP8A1 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, CDC50a

ProteinName: CDC50a / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 4 mg/mL / pH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 64 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 138711
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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