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- EMDB-25838: Cryo-EM structure of nanodisc-embedded human ABCA1 -

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Basic information

Entry
Database: EMDB / ID: EMD-25838
TitleCryo-EM structure of nanodisc-embedded human ABCA1
Map data
Sample
  • Organelle or cellular component: Human ABCA1 reconstituted into nanodiscs
    • Protein or peptide: Phospholipid-transporting ATPase ABCA1
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 / platelet dense granule organization / intracellular cholesterol transport ...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 / platelet dense granule organization / intracellular cholesterol transport / high-density lipoprotein particle binding / phospholipid transporter activity / protein transmembrane transport / response to laminar fluid shear stress / floppase activity / HDL assembly / phosphatidylserine floppase activity / cellular response to cholesterol / peptide secretion / phosphatidylcholine floppase activity / phospholipid homeostasis / phosphatidylcholine binding / phospholipid efflux / export across plasma membrane / reverse cholesterol transport / cholesterol transfer activity / high-density lipoprotein particle assembly / lipoprotein biosynthetic process / P-type phospholipid transporter / regulation of Cdc42 protein signal transduction / phospholipid translocation / syntaxin binding / cholesterol efflux / endosomal transport / phagocytosis, engulfment / intracellular vesicle / lysosome organization / cholesterol binding / negative regulation of cholesterol storage / cellular response to cytokine stimulus / apolipoprotein binding / negative regulation of macrophage derived foam cell differentiation / cellular response to low-density lipoprotein particle stimulus / protein secretion / endocytic vesicle / ATPase-coupled transmembrane transporter activity / protein transmembrane transporter activity / positive regulation of cholesterol efflux / ABC-type transporter activity / cellular response to retinoic acid / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / phagocytic vesicle / 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 / intracellular membrane-bounded organelle / signaling receptor binding / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsPlummer AM / Culbertson AT / Morales-Perez CL / Liao M
Funding support United States, 3 items
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, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateApr 26, 2023-
Current statusApr 26, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25838.png

Downloads & links

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Map

FileDownload / File: emd_25838.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy AUTHOR: 0.0176
Minimum - Maximum-0.060757667 - 0.11828143
Average (Standard dev.)0.00016269287 (±0.004347093)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 317.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human ABCA1 reconstituted into nanodiscs

EntireName: Human ABCA1 reconstituted into nanodiscs
Components
  • Organelle or cellular component: Human ABCA1 reconstituted into nanodiscs
    • Protein or peptide: Phospholipid-transporting ATPase ABCA1

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Supramolecule #1: Human ABCA1 reconstituted into nanodiscs

SupramoleculeName: Human ABCA1 reconstituted into nanodiscs / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Phospholipid-transporting ATPase ABCA1

MacromoleculeName: Phospholipid-transporting ATPase ABCA1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 255.391797 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GGGASEFVDM ACWPQLRLLL WKNLTFRRRQ TCQLLLEVAW PLFIFLILIS VRLSYPPYEQ HECHFPNKAM PSAGTLPWVQ GIICNANNP CFRYPTPGEA PGVVGNFNKS IVARLFSDAR RLLLYSQKDT SMKDMRKVLR TLQQIKKSSS NLKLQDFLVD N ETFSGFLY ...String:
GGGASEFVDM ACWPQLRLLL WKNLTFRRRQ TCQLLLEVAW PLFIFLILIS VRLSYPPYEQ HECHFPNKAM PSAGTLPWVQ GIICNANNP CFRYPTPGEA PGVVGNFNKS IVARLFSDAR RLLLYSQKDT SMKDMRKVLR TLQQIKKSSS NLKLQDFLVD N ETFSGFLY HNLSLPKSTV DKMLRADVIL HKVFLQGYQL HLTSLCNGSK SEEMIQLGDQ EVSELCGLPR EKLAAAERVL RS NMDILKP ILRTLNSTSP FPSKELAEAT KTLLHSLGTL AQELFSMRSW SDMRQEVMFL TNVNSSSSST QIYQAVSRIV CGH PEGGGL KIKSLNWYED NNYKALFGGN GTEEDAETFY DNSTTPYCND LMKNLESSPL SRIIWKALKP LLVGKILYTP DTPA TRQVM AEVNKTFQEL AVFHDLEGMW EELSPKIWTF MENSQEMDLV RMLLDSRDND HFWEQQLDGL DWTAQDIVAF LAKHP EDVQ SSNGSVYTWR EAFNETNQAI RTISRFMECV NLNKLEPIAT EVWLINKSME LLDERKFWAG IVFTGITPGS IELPHH VKY KIRMDIDNVE RTNKIKDGYW DPGPRADPFE DMRYVWGGFA YLQDVVEQAI IRVLTGTEKK TGVYMQQMPY PCYVDDI FL RVMSRSMPLF MTLAWIYSVA VIIKGIVYEK EARLKETMRI MGLDNSILWF SWFISSLIPL LVSAGLLVVI LKLGNLLP Y SDPSVVFVFL SVFAVVTILQ CFLISTLFSR ANLAAACGGI IYFTLYLPYV LCVAWQDYVG FTLKIFASLL SPVAFGFGC EYFALFEEQG IGVQWDNLFE SPVEEDGFNL TTSVSMMLFD TFLYGVMTWY IEAVFPGQYG IPRPWYFPCT KSYWFGEESD EKSHPGSNQ KRISEICMEE EPTHLKLGVS IQNLVKVYRD GMKVAVDGLA LNFYEGQITS FLGHNGAGKT TTMSILTGLF P PTSGTAYI LGKDIRSEMS TIRQNLGVCP QHNVLFDMLT VEEHIWFYAR LKGLSEKHVK AEMEQMALDV GLPSSKLKSK TS QLSGGMQ RKLSVALAFV GGSKVVILDE PTAGVDPYSR RGIWELLLKY RQGRTIILST HHMDEADVLG DRIAIISHGK LCC VGSSLF LKNQLGTGYY LTLVKKDVES SLSSCRNSSS TVSYLKKEDS VSQSSSDAGL GSDHESDTLT IDVSAISNLI RKHV SEARL VEDIGHELTY VLPYEAAKEG AFVELFHEID DRLSDLGISS YGISETTLEE IFLKVAEESG VDAETSDGTL PARRN RRAF GDKQSCLRPF TEDDAADPND SDIDPESRET DLLSGMDGKG SYQVKGWKLT QQQFVALLWK RLLIARRSRK GFFAQI VLP AVFVCIALVF SLIVPPFGKY PSLELQPWMY NEQYTFVSND APEDTGTLEL LNALTKDPGF GTRCMEGNPI PDTPCQA GE EEWTTAPVPQ TIMDLFQNGN WTMQNPSPAC QCSSDKIKKM LPVCPPGAGG LPPPQRKQNT ADILQDLTGR NISDYLVK T YVQIIAKSLK NKIWVNEFRY GGFSLGVSNT QALPPSQEVN DAIKQMKKHL KLAKDSSADR FLNSLGRFMT GLDTKNNVK VWFNNKGWHA ISSFLNVINN AILRANLQKG ENPSHYGITA FNHPLNLTKQ QLSEVALMTT SVDVLVSICV IFAMSFVPAS FVVFLIQER VSKAKHLQFI SGVKPVIYWL SNFVWDMCNY VVPATLVIII FICFQQKSYV SSTNLPVLAL LLLLYGWSIT P LMYPASFV FKIPSTAYVV LTSVNLFIGI NGSVATFVLE LFTDNKLNNI NDILKSVFLI FPHFCLGRGL IDMVKNQAMA DA LERFGEN RFVSPLSWDL VGRNLFAMAV EGVVFFLITV LIQYRFFIRP RPVNAKLSPL NDEDEDVRRE RQRILDGGGQ NDI LEIKEL TKIYRRKRKP AVDRICVGIP PGECFGLLGV NGAGKSSTFK MLTGDTTVTR GDAFLNKNSI LSNIHEVHQN MGYC PQFDA ITELLTGREH VEFFALLRGV PEKEVGKVGE WAIRKLGLVK YGEKYAGNYS GGNKRKLSTA MALIGGPPVV FLDEP TTGM DPKARRFLWN CALSVVKEGR SVVLTSHSME ECEALCTRMA IMVNGRFRCL GSVQHLKNRF GDGYTIVVRI AGSNPD LKP VQDFFGLAFP GSVLKEKHRN MLQYQLPSSL SSLARIFSIL SQSKKRLHIE DYSVSQTTLD QVFVNFAKDQ SDDDHLK DL SLHKNQTVVD VAVLTSFLQD EKVKESYV

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
100.0 mMNaClSodium chloride
5.0 %C3H8O3Glycerol
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE
DetailsHomogeneous, monodisperse sample of ABCA1 in nanodiscs

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Image recordingImage recording ID: 1 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.04 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #1~

Microscopy ID1
MicroscopeFEI TALOS ARCTICA
Image recordingImage recording ID: 2 / Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.07 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 36000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Image recording ID1
Particle selectionNumber selected: 364684
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5xjy


Details: Also used 7ROQ
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Number images used: 32360
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.7)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5xjy


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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