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- EMDB-27079: Minor conformation of nanodisc embedded ABCA1 -

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Basic information

Entry
Database: EMDB / ID: EMD-27079
TitleMinor conformation of nanodisc embedded ABCA1
Map data
Sample
  • Complex: Human ABCA1 reconstituted into nanodiscs
    • Protein or peptide: ABCA1 in nanodisc
Keywordssterol transport / ABC transporter / phospholipid transport / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 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
DepositionMay 23, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27079.png

Downloads & links

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Map

FileDownload / File: emd_27079.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: 0.0168
Minimum - Maximum-0.015572204 - 0.054102495
Average (Standard dev.)0.00012381654 (±0.002375911)
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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Half map: #2

Fileemd_27079_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27079_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human ABCA1 reconstituted into nanodiscs

EntireName: Human ABCA1 reconstituted into nanodiscs
Components
  • Complex: Human ABCA1 reconstituted into nanodiscs
    • Protein or peptide: ABCA1 in nanodisc

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Supramolecule #1: Human ABCA1 reconstituted into nanodiscs

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

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Macromolecule #1: ABCA1 in nanodisc

MacromoleculeName: ABCA1 in nanodisc / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GGGASEFVDM ACWPQLRLLL WKNLTFRRRQ TCQLLLEVAW PLFIFLILIS VRLSYPPYEQ HECHFPNKA MPSAGTLPWV QGIICNANNP CFRYPTPGEA PGVVGNFNKS IVARLFSDAR RLLLYSQKDT SMKDMRKVL RTLQQIKKSS SNLKLQDFLV DNETFSGFLY ...String:
GGGASEFVDM ACWPQLRLLL WKNLTFRRRQ TCQLLLEVAW PLFIFLILIS VRLSYPPYEQ HECHFPNKA MPSAGTLPWV QGIICNANNP CFRYPTPGEA PGVVGNFNKS IVARLFSDAR RLLLYSQKDT SMKDMRKVL RTLQQIKKSS SNLKLQDFLV DNETFSGFLY HNLSLPKSTV DKMLRADVIL H KVFLQGYQ LHLTSLCNGS KSEEMIQLGD QEVSELCGLP REKLAAAERV LRSNMDILKP IL RTLNSTS PFPSKELAEA TKTLLHSLGT LAQELFSMRS WSDMRQEVMF LTNVNSSSSS TQI YQAVSR IVCGHPEGGG LKIKSLNWYE DNNYKALFGG NGTEEDAETF YDNSTTPYCN DLMK NLESS PLSRIIWKAL KPLLVGKILY TPDTPATRQV MAEVNKTFQE LAVFHDLEGM WEELS PKIW TFMENSQEMD LVRMLLDSRD NDHFWEQQLD GLDWTAQDIV AFLAKHPEDV QSSNGS VYT WREAFNETNQ AIRTISRFME CVNLNKLEPI ATEVWLINKS MELLDERKFW AGIVFTG IT PGSIELPHHV KYKIRMDIDN VERTNKIKDG YWDPGPRADP FEDMRYVWGG FAYLQDVV E QAIIRVLTGT EKKTGVYMQQ MPYPCYVDDI FLRVMSRSMP LFMTLAWIYS VAVIIKGIV YEKEARLKET MRIMGLDNSI LWFSWFISSL IPLLVSAGLL VVILKLGNLL PYSDPSVVFV FLSVFAVVT ILQCFLISTL FSRANLAAAC GGIIYFTLYL PYVLCVAWQD YVGFTLKIFA S LLSPVAFG FGCEYFALFE EQGIGVQWDN LFESPVEEDG FNLTTSVSMM LFDTFLYGVM TW YIEAVFP GQYGIPRPWY FPCTKSYWFG EESDEKSHPG SNQKRISEIC MEEEPTHLKL GVS IQNLVK VYRDGMKVAV DGLALNFYEG QITSFLGHNG AGKTTTMSIL TGLFPPTSGT AYIL GKDIR SEMSTIRQNL GVCPQHNVLF DMLTVEEHIW FYARLKGLSE KHVKAEMEQM ALDVG LPSS KLKSKTSQLS GGMQRKLSVA LAFVGGSKVV ILDEPTAGVD PYSRRGIWEL LLKYRQ GRT IILSTHHMDE ADVLGDRIAI ISHGKLCCVG SSLFLKNQLG TGYYLTLVKK DVESSLS SC RNSSSTVSYL KKEDSVSQSS SDAGLGSDHE SDTLTIDVSA ISNLIRKHVS EARLVEDI G HELTYVLPYE AAKEGAFVEL FHEIDDRLSD LGISSYGISE TTLEEIFLKV AEESGVDAE TSDGTLPARR NRRAFGDKQS CLRPFTEDDA ADPNDSDIDP ESRETDLLSG MDGKGSYQVK GWKLTQQQF VALLWKRLLI ARRSRKGFFA QIVLPAVFVC IALVFSLIVP PFGKYPSLEL Q PWMYNEQY TFVSNDAPED TGTLELLNAL TKDPGFGTRC MEGNPIPDTP CQAGEEEWTT AP VPQTIMD LFQNGNWTMQ NPSPACQCSS DKIKKMLPVC PPGAGGLPPP QRKQNTADIL QDL TGRNIS DYLVKTYVQI IAKSLKNKIW VNEFRYGGFS LGVSNTQALP PSQEVNDAIK QMKK HLKLA KDSSADRFLN SLGRFMTGLD TKNNVKVWFN NKGWHAISSF LNVINNAILR ANLQK GENP SHYGITAFNH PLNLTKQQLS EVALMTTSVD VLVSICVIFA MSFVPASFVV FLIQER VSK AKHLQFISGV KPVIYWLSNF VWDMCNYVVP ATLVIIIFIC FQQKSYVSST NLPVLAL LL LLYGWSITPL MYPASFVFKI PSTAYVVLTS VNLFIGINGS VATFVLELFT DNKLNNIN D ILKSVFLIFP HFCLGRGLID MVKNQAMADA LERFGENRFV SPLSWDLVGR NLFAMAVEG VVFFLITVLI QYRFFIRPRP VNAKLSPLND EDEDVRRERQ RILDGGGQND ILEIKELTKI YRRKRKPAV DRICVGIPPG ECFGLLGVNG AGKSSTFKML TGDTTVTRGD AFLNKNSILS N IHEVHQNM GYCPQFDAIT ELLTGREHVE FFALLRGVPE KEVGKVGEWA IRKLGLVKYG EK YAGNYSG GNKRKLSTAM ALIGGPPVVF LDEPTTGMDP KARRFLWNCA LSVVKEGRSV VLT SHSMEE CEALCTRMAI MVNGRFRCLG SVQHLKNRFG DGYTIVVRIA GSNPDLKPVQ DFFG LAFPG SVLKEKHRNM LQYQLPSSLS SLARIFSILS QSKKRLHIED YSVSQTTLDQ VFVNF AKDQ SDDDHLKDLS LHKNQTVVDV AVLTSFLQDE KVKESYV

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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
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 reconstructionResolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Number images used: 26400
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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