Journal: 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
Deposition
Jun 7, 2019
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Header (metadata) release
Aug 28, 2019
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Map release
Aug 28, 2019
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Update
Nov 13, 2019
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Current status
Nov 13, 2019
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
EMPIAR-10303 (Title: Cryo-EM structures of human P4-ATPase flippase / Data size: 11.0 TB Data #1: Unaligned movies for E2P class 1,2,3 [micrographs - multiframe] Data #2: Unaligned movies for E2Pi-PL and E1P [micrographs - multiframe] Data #3: Unaligned movies for E1P-ADP [micrographs - multiframe] Data #4: Unaligned movies for E1 class1,2,3 [micrographs - multiframe] Data #5: Unaligned movies for E1-ATP class 1,2,3 [micrographs - multiframe])
Expression System: Homo sapiens (human) / Vector: pcDNA3.4
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Component #2: protein, ATP8A1
Protein
Name: ATP8A1 / Recombinant expression: No
Source
Species: Homo sapiens (human)
Source (engineered)
Expression System: Homo sapiens (human)
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Component #3: protein, CDC50a
Protein
Name: CDC50a / Recombinant expression: No
Source
Species: Homo sapiens (human)
Source (engineered)
Expression System: Homo sapiens (human)
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Experimental details
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Sample preparation
Specimen
Specimen state: Particle / Method: cryo EM
Sample solution
Specimen conc.: 4 mg/mL / pH: 7.5
Vitrification
Cryogen name: ETHANE
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Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Imaging
Microscope: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 64 e/Å2 / Illumination mode: OTHER
Lens
Imaging mode: BRIGHT FIELD
Specimen Holder
Model: OTHER
Camera
Detector: OTHER
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Image processing
Processing
Method: single particle reconstruction / Number of projections: 138711
3D reconstruction
Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)
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