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- PDB-8eeb: Cryo-EM structure of human ABCA7 in Digitonin -

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Basic information

Entry
Database: PDB / ID: 8eeb
TitleCryo-EM structure of human ABCA7 in Digitonin
ComponentsPhospholipid-transporting ATPase ABCA7
KeywordsTRANSPORT PROTEIN / ABC transporter / Lipid Transporter / ABC exporter
Function / homology
Function and homology information


plasma membrane raft organization / apolipoprotein A-I receptor activity / positive regulation of engulfment of apoptotic cell / phospholipid transporter activity / positive regulation of phospholipid efflux / negative regulation of amyloid precursor protein biosynthetic process / ABC transporters in lipid homeostasis / phospholipid efflux / floppase activity / phosphatidylserine floppase activity ...plasma membrane raft organization / apolipoprotein A-I receptor activity / positive regulation of engulfment of apoptotic cell / phospholipid transporter activity / positive regulation of phospholipid efflux / negative regulation of amyloid precursor protein biosynthetic process / ABC transporters in lipid homeostasis / phospholipid efflux / floppase activity / phosphatidylserine floppase activity / high-density lipoprotein particle assembly / positive regulation of amyloid-beta clearance / amyloid-beta clearance by cellular catabolic process / peptide cross-linking / phosphatidylcholine floppase activity / negative regulation of endocytosis / positive regulation of protein localization to cell surface / regulation of amyloid precursor protein catabolic process / P-type phospholipid transporter / cholesterol efflux / apolipoprotein A-I-mediated signaling pathway / phospholipid translocation / phagocytic cup / negative regulation of amyloid-beta formation / negative regulation of MAPK cascade / negative regulation of PERK-mediated unfolded protein response / glial cell projection / amyloid-beta formation / positive regulation of cholesterol efflux / regulation of lipid metabolic process / protein localization to nucleus / ATPase-coupled transmembrane transporter activity / ABC-type transporter activity / positive regulation of phagocytosis / phagocytosis / visual learning / memory / ruffle membrane / cell junction / early endosome membrane / positive regulation of ERK1 and ERK2 cascade / Golgi membrane / intracellular membrane-bounded organelle / Golgi apparatus / cell surface / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
ABC-2 family transporter protein / ABC transporter A / 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
Phospholipid-transporting ATPase ABCA7
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsAlam, A. / Le, L.T.M. / Thompson, J.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)1R21AG069180 United States
CitationJournal: EMBO J / Year: 2023
Title: Cryo-EM structures of human ABCA7 provide insights into its phospholipid translocation mechanisms.
Authors: Le Thi My Le / James Robert Thompson / Sepehr Dehghani-Ghahnaviyeh / Shashank Pant / Phuoc Xuan Dang / Jarrod Bradley French / Takahisa Kanikeyo / Emad Tajkhorshid / Amer Alam /
Abstract: Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly ...Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly resolved at the molecular level. Here we report cryo-EM structures of lipid-embedded human ABCA7 in an open state and in a nucleotide-bound, closed state at resolutions between 3.6 and 4.0 Å. The former reveals an ordered patch of bilayer lipids traversing the transmembrane domain (TMD), while the latter reveals a lipid-free, closed TMD with a small extracellular opening. These structures offer a structural framework for both substrate entry and exit from the ABCA7 TMD and highlight conserved rigid-body motions that underlie the associated conformational transitions. Combined with functional analysis and molecular dynamics (MD) simulations, our data also shed light on lipid partitioning into the ABCA7 TMD and localized membrane perturbations that underlie ABCA7 function and have broader implications for other ABCA family transporters.
History
DepositionSep 6, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 15, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phospholipid-transporting ATPase ABCA7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)237,93010
Polymers234,5991
Non-polymers3,3319
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phospholipid-transporting ATPase ABCA7 / ABCA-SSN / ATP-binding cassette sub-family A member 7 / Autoantigen SS-N / Macrophage ABC transporter


Mass: 234598.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABCA7 / Cell line (production host): HEK293TREX / Production host: Homo sapiens (human)
References: UniProt: Q8IZY2, P-type phospholipid transporter
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-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.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: 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.: 4 / Source method: obtained synthetically / 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
Has ligand of interestN

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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 ABCA7 in Digitonin / Type: COMPLEX
Details: Human ABCA7 recombinantly expressed in HEK293 TREX cell line and reconstituted in Digitonin
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.234350 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5 / Details: 25mM HEPES pH 7.5, 150mM NaCl, 0.035% Digitonin
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Human ABCA7 in Digitonin
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Image recordingAverage exposure time: 60 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3.1/4.0initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.1/4.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149590 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6JBJ

6jbj

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215021
ELECTRON MICROSCOPYf_angle_d0.62620430
ELECTRON MICROSCOPYf_dihedral_angle_d13.8585482
ELECTRON MICROSCOPYf_chiral_restr0.0452368
ELECTRON MICROSCOPYf_plane_restr0.0042607

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