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- EMDB-21441: ABCG2 in the apo-closed conformation in the presence of SN38 -

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Basic information

Entry
Database: EMDB / ID: EMD-21441
TitleABCG2 in the apo-closed conformation in the presence of SN38
Map dataCryo-EM map of ABCG2 in the apo-closed conformation in the presence of SN38
Sample
  • Complex: ABCG2 reconstituted in lipid nanodisc
    • Protein or peptide: ABCG2
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsOrlando BJ / Maofu L
Funding support United States, 1 items
OrganizationGrant numberCountry
American Cancer SocietyPF1721201DMC United States
CitationJournal: Nat Commun / Year: 2020
Title: ABCG2 transports anticancer drugs via a closed-to-open switch.
Authors: Benjamin J Orlando / Maofu Liao /
Abstract: ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer ...ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer drug bound to ABCG2 has been resolved, and the mechanisms of multidrug transport remain obscure. Such a gap of knowledge limits the development of novel compounds that block or evade this critical molecular pump. Here we present single-particle cryo-EM studies of ABCG2 in the apo state, and bound to the three structurally distinct chemotherapeutics. Without the binding of conformation-selective antibody fragments or inhibitors, the resting ABCG2 adopts a closed conformation. Our cryo-EM, biochemical, and functional analyses reveal the binding mode of three chemotherapeutic compounds, demonstrate how these molecules open the closed conformation of the transporter, and establish that imatinib is particularly effective in stabilizing the inward facing conformation of ABCG2. Together these studies reveal the previously unrecognized conformational cycle of ABCG2.
History
DepositionFeb 22, 2020-
Header (metadata) releaseMar 25, 2020-
Map releaseMay 13, 2020-
UpdateMay 20, 2020-
Current statusMay 20, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21441.png

Downloads & links

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Map

FileDownload / File: emd_21441.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of ABCG2 in the apo-closed conformation in the presence of SN38
Voxel sizeX=Y=Z: 1.23 Å
Density
Contour LevelBy AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum-0.16759709 - 0.27397165
Average (Standard dev.)-0.00008790960 (±0.0131454)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 236.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z236.160236.160236.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.1680.274-0.000

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Supplemental data

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Additional map: Unfiltered and unsharpened cryo-EM map of ABCG2 in...

Fileemd_21441_additional.map
AnnotationUnfiltered and unsharpened cryo-EM map of ABCG2 in the apo-closed conformation in the presence of SN38
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ABCG2 reconstituted in lipid nanodisc

EntireName: ABCG2 reconstituted in lipid nanodisc
Components
  • Complex: ABCG2 reconstituted in lipid nanodisc
    • Protein or peptide: ABCG2

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Supramolecule #1: ABCG2 reconstituted in lipid nanodisc

SupramoleculeName: ABCG2 reconstituted in lipid nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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Macromolecule #1: ABCG2

MacromoleculeName: ABCG2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNING IMKPGLNAIL GPTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC N SGYVVQDD VVMGTLTVRE NLQFSAALRL ATTMTNHEKN ERINRVIQEL ...String:
MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNING IMKPGLNAIL GPTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC N SGYVVQDD VVMGTLTVRE NLQFSAALRL ATTMTNHEKN ERINRVIQEL GLDKVADSKV GT QFIRGVS GGERKRTSIG MELITDPSIL FLDEPTTGLD SSTANAVLLL LKRMSKQGRT IIF SIHQPR YSIFKLFDSL TLLASGRLMF HGPAQEALGY FESAGYHCEA YNNPADFFLD IING DSTAV ALNREEDFKA TEIIEPSKQD KPLIEKLAEI YVNSSFYKET KAELHQLSGG EKKKK ITVF KEISYTTSFC HQLRWVSKRS FKNLLGNPQA SIAQIIVTVV LGLVIGAIYF GLKNDS TGI QNRAGVLFFL TTNQCFSSVS AVELFVVEKK LFIHEYISGY YRVSSYFLGK LLSDLLP MR MLPSIIFTCI VYFMLGLKPK ADAFFVMMFT LMMVAYSASS MALAIAAGQS VVSVATLL M TICFVFMMIF SGLLVNLTTI ASWLSWLQYF SIPRYGFTAL QHNEFLGQNF CPGLNATGN NPCNYATCTG EEYLVKQGID LSPWGLWKNH VALACMIVIF LTIAYLKLLF LKKYS

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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

Concentration1.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
25.0 mMC4H11NO3Tris base
GridModel: Quantifoil R1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 92 % / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 31000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 8.0 sec. / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: Relion
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 86755
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL

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