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- PDB-7bsq: Cryo-EM structure of a human ATP11C-CDC50A flippase in E1AlF-ADP state -

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Basic information

Entry
Database: PDB / ID: 7bsq
TitleCryo-EM structure of a human ATP11C-CDC50A flippase in E1AlF-ADP state
Components
  • ATP11C
  • CDC50A
KeywordsMEMBRANE PROTEIN / Flippase / P-type ATPase / P4-ATPase / Phospholipid
Function / homology
Function and homology information


positive regulation of phospholipid translocation / aminophospholipid flippase activity / aminophospholipid transport / phosphatidylserine flippase activity / protein localization to endosome / phosphatidylserine floppase activity / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / positive regulation of protein exit from endoplasmic reticulum / xenobiotic transmembrane transport ...positive regulation of phospholipid translocation / aminophospholipid flippase activity / aminophospholipid transport / phosphatidylserine flippase activity / protein localization to endosome / phosphatidylserine floppase activity / phospholipid-translocating ATPase complex / ATPase-coupled intramembrane lipid transporter activity / positive regulation of protein exit from endoplasmic reticulum / xenobiotic transmembrane transport / phosphatidylethanolamine flippase activity / P-type phospholipid transporter / phospholipid translocation / azurophil granule membrane / transport vesicle membrane / Ion transport by P-type ATPases / specific granule membrane / monoatomic ion transmembrane transport / positive regulation of neuron projection development / recycling endosome / recycling endosome membrane / late endosome membrane / early endosome membrane / apical plasma membrane / lysosomal membrane / Neutrophil degranulation / endoplasmic reticulum membrane / structural molecule activity / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain ...CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / TETRAFLUOROALUMINATE ION / Phospholipid-transporting ATPase IG / Cell cycle control protein 50A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAbe, K. / Nishizawa, T. / Nakanishi, H.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR14M4 Japan
Japan Agency for Medical Research and Development (AMED) Japan
Japan Society for the Promotion of Science (JSPS)17H03653 Japan
CitationJournal: Cell Rep / Year: 2020
Title: Transport Cycle of Plasma Membrane Flippase ATP11C by Cryo-EM.
Authors: Hanayo Nakanishi / Tomohiro Nishizawa / Katsumori Segawa / Osamu Nureki / Yoshinori Fujiyoshi / Shigekazu Nagata / Kazuhiro Abe /
Abstract: ATP11C, a plasma membrane phospholipid flippase, maintains the asymmetric distribution of phosphatidylserine accumulated in the inner leaflet. Caspase-dependent inactivation of ATP11C is essential ...ATP11C, a plasma membrane phospholipid flippase, maintains the asymmetric distribution of phosphatidylserine accumulated in the inner leaflet. Caspase-dependent inactivation of ATP11C is essential for an apoptotic "eat me" signal, phosphatidylserine exposure, which prompts phagocytes to engulf cells. We show six cryo-EM structures of ATP11C at 3.0-4.0 Å resolution in five different states of the transport cycle. A structural comparison reveals phosphorylation-driven domain movements coupled with phospholipid binding. Three structures of phospholipid-bound states visualize phospholipid translocation accompanied by the rearrangement of transmembrane helices and an unwound portion at the occlusion site, and thus they detail the basis for head group recognition and the locality of the protein-bound acyl chains in transmembrane grooves. Invariant Lys880 and the surrounding hydrogen-bond network serve as a pivot point for helix bending and precise P domain inclination, which is crucial for dephosphorylation. The structures detail key features of phospholipid translocation by ATP11C, and a common basic mechanism for flippases is emerging.
History
DepositionMar 31, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 30, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 14, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Assembly

Deposited unit
A: ATP11C
C: CDC50A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,9129
Polymers165,3042
Non-polymers1,6087
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10670 Å2
ΔGint-40 kcal/mol
Surface area56580 Å2

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Components

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Protein , 2 types, 2 molecules AC

#1: Protein ATP11C /


Mass: 124403.617 Da / Num. of mol.: 1 / Mutation: delete 7 a.a. at N-term and 38 a.a. at C-term
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q8NB49*PLUS
#2: Protein CDC50A


Mass: 40900.801 Da / Num. of mol.: 1 / Mutation: N180Q, S292W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q9NV96*PLUS

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Sugars , 2 types, 3 molecules

#3: Polysaccharide alpha-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...alpha-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2/a4-b1_b3-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(3+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#7: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 4 molecules

#4: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Sequence detailsNCBI Reference Sequence accession IDs are NCBI XM_005262405.1 for ATP11C and NP_001340741.2 for CDC50A.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: human ATP11C-CDC50A flippase / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293
Buffer solutionpH: 6.5
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 500000 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 54.07 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.013711280
ELECTRON MICROSCOPYf_angle_d0.938415295
ELECTRON MICROSCOPYf_chiral_restr0.12611728
ELECTRON MICROSCOPYf_plane_restr0.00561920
ELECTRON MICROSCOPYf_dihedral_angle_d22.12044146

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