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- PDB-7tdt: Cryo-EM structure of nanodisc-embedded human ABCA1 -

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Basic information

Entry
Database: PDB / ID: 7tdt
TitleCryo-EM structure of nanodisc-embedded human ABCA1
ComponentsPhospholipid-transporting ATPase ABCA1
KeywordsMEMBRANE PROTEIN / sterol transport / ABC transporter / phospholipid transport
Function / homology
Function and homology information


signal release / sphingolipid floppase activity / regulation of high-density lipoprotein particle assembly / apolipoprotein A-I receptor activity / positive regulation of high-density lipoprotein particle assembly / Defective ABCA1 causes TGD / response to vitamin B3 / apolipoprotein A-I binding / intracellular cholesterol transport / platelet dense granule organization ...signal release / sphingolipid floppase activity / regulation of high-density lipoprotein particle assembly / apolipoprotein A-I receptor activity / positive regulation of high-density lipoprotein particle assembly / Defective ABCA1 causes TGD / response to vitamin B3 / apolipoprotein A-I binding / intracellular cholesterol transport / platelet dense granule organization / phospholipid transporter activity / high-density lipoprotein particle binding / protein transmembrane transport / response to laminar fluid shear stress / phospholipid efflux / floppase activity / HDL assembly / peptide secretion / cholesterol transfer activity / reverse cholesterol transport / phosphatidylserine floppase activity / high-density lipoprotein particle assembly / lipoprotein biosynthetic process / cellular response to cholesterol / phospholipid homeostasis / phosphatidylcholine floppase activity / phosphatidylcholine binding / regulation of Cdc42 protein signal transduction / export across plasma membrane / P-type phospholipid transporter / cholesterol efflux / phospholipid translocation / syntaxin binding / cholesterol binding / endosomal transport / phagocytosis, engulfment / lysosome organization / intracellular vesicle / negative regulation of cholesterol storage / cellular response to cytokine stimulus / apolipoprotein binding / positive regulation of cholesterol efflux / negative regulation of macrophage derived foam cell differentiation / cellular response to low-density lipoprotein particle stimulus / protein transmembrane transporter activity / endocytic vesicle / protein secretion / ATPase-coupled transmembrane transporter activity / ABC-type transporter activity / phagocytic vesicle / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / cellular response to retinoic acid / cholesterol metabolic process / cholesterol homeostasis / adenylate cyclase-activating G protein-coupled receptor signaling pathway / PPARA activates gene expression / small GTPase binding / cellular response to xenobiotic stimulus / ATPase binding / basolateral plasma membrane / cellular response to lipopolysaccharide / endosome / membrane raft / G protein-coupled receptor signaling pathway / external side of plasma membrane / signaling receptor binding / intracellular membrane-bounded organelle / endoplasmic reticulum membrane / perinuclear region of cytoplasm / Golgi apparatus / 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 ABCA1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsPlummer, A.M. / Culbertson, A.T. / Morales-Perez, C.L. / Liao, M.
Funding support United States, 3items
OrganizationGrant numberCountry
American Heart Association18POST34030341 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1F32GM136092-01 United States
American Cancer SocietyPF-20-107-01-TBE United States
CitationJournal: J Mol Biol / Year: 2023
Title: Activity and Structural Dynamics of Human ABCA1 in a Lipid Membrane.
Authors: Ashlee M Plummer-Medeiros / Alan T Culbertson / Claudio L Morales-Perez / Maofu Liao /
Abstract: The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular ...The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer's disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing. In this work, we established a stable expression system for both a human cell-based sterol export assay and protein purification for in vitro biochemical and structural studies. ABCA1 produced in this system was active in sterol export and displayed enhanced ATPase activity after reconstitution into a lipid bilayer. Our single-particle cryo-EM study of ABCA1 in nanodiscs showed protein induced membrane curvature, revealed multiple distinct conformations, and generated a structure of nanodisc-embedded ABCA1 at 4.0-Å resolution representing a previously unknown conformation. Comparison of different ABCA1 structures and molecular dynamics simulations demonstrates both concerted domain movements and conformational variations within each domain. Taken together, our platform for producing and characterizing ABCA1 in a lipid membrane enabled us to gain important mechanistic and structural insights and paves the way for investigating modulators that target the functions of ABCA1.
History
DepositionJan 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phospholipid-transporting ATPase ABCA1


Theoretical massNumber of molelcules
Total (without water)255,3921
Polymers255,3921
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phospholipid-transporting ATPase ABCA1 / ATP-binding cassette sub-family A member 1 / ATP-binding cassette transporter 1 / ABC-1 / ATP- ...ATP-binding cassette sub-family A member 1 / ATP-binding cassette transporter 1 / ABC-1 / ATP-binding cassette 1 / Cholesterol efflux regulatory protein


Mass: 255391.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABCA1, ABC1, CERP / Production host: Homo sapiens (human)
References: UniProt: O95477, P-type phospholipid transporter

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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 ABCA1 reconstituted into nanodiscs / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2100 mMSodium chlorideNaClSodium chloride1
35 %GlycerolC3H8O31
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Homogeneous, monodisperse sample of ABCA1 in nanodiscs
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company /
Model: Talos Arctica / Image courtesy: FEI Company
EM imaging

Alignment procedure: COMA FREE / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Specimen-ID: 1

IDAccelerating voltage (kV)C2 aperture diameter (µm)CryogenModelNominal defocus max (nm)Nominal defocus min (nm)Nominal magnification (X)Specimen holder model
130050NITROGENFEI TITAN KRIOS25001000105000FEI TITAN KRIOS AUTOGRID HOLDER
2200FEI TALOS ARCTICA3500150036000
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
111.04GATAN K3 BIOQUANTUM (6k x 4k)
221.07GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryImaging-ID
2SerialEMimage acquisition1
4CTFFINDCTF correction
7Coot0.9.5model fitting
9PHENIX1.19.2model refinement
10SerialEMimage acquisition2
11RELION3.0.7initial Euler assignment
12RELION3.0.7final Euler assignment
13RELION3.0.7classification
14RELION3.0.73D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 364684
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32360 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5XJY

5xjy


Pdb chain-ID: A
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314783
ELECTRON MICROSCOPYf_angle_d0.62820049
ELECTRON MICROSCOPYf_dihedral_angle_d4.5071943
ELECTRON MICROSCOPYf_chiral_restr0.0392277
ELECTRON MICROSCOPYf_plane_restr0.0042521

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