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- EMDB-12952: ABCG2 topotecan turnover-2 state -

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Basic information

Entry
Database: EMDB / ID: EMD-12952
TitleABCG2 topotecan turnover-2 state
Map data
Sample
  • Complex: ABCG2 in complex with topotecan under turnover condition
    • Protein or peptide: Broad substrate specificity ATP-binding cassette transporter ABCG2
  • Ligand: (S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3',4':6,7]INOLIZINO[1,2-B]-QUINOLINE-3,14(4H,12H)-DIONE
  • Ligand: CHOLESTEROL
  • Ligand: DIUNDECYL PHOSPHATIDYL CHOLINE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Function / homology
Function and homology information


biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / sphingolipid transporter activity / renal urate salt excretion / Abacavir transmembrane transport / urate metabolic process / urate transmembrane transporter activity / external side of apical plasma membrane ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / sphingolipid transporter activity / renal urate salt excretion / Abacavir transmembrane transport / urate metabolic process / urate transmembrane transporter activity / external side of apical plasma membrane / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / organic anion transport / organic anion transmembrane transporter activity / xenobiotic transport across blood-brain barrier / transepithelial transport / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / Ciprofloxacin ADME / NFE2L2 regulating MDR associated enzymes / ABC-type xenobiotic transporter activity / Differentiation of keratinocytes in interfollicular epidermis in mammalian skin / cellular detoxification / Heme biosynthesis / Heme degradation / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / transport across blood-brain barrier / Iron uptake and transport / mitochondrial membrane / brush border membrane / transmembrane transport / membrane raft / apical plasma membrane / protein homodimerization activity / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / plasma membrane
Similarity search - Function
ABC transporter family G domain / ABC-2 type transporter / : / ABC-2 type transporter / ABC-2 type transporter / 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
Broad substrate specificity ATP-binding cassette transporter ABCG2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsYu Q / Ni D / Kowal J / Manolaridis I / Jackson SM / Stahlberg H / Locher KP
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundationnot applicable Switzerland
CitationJournal: Nat Commun / Year: 2021
Title: Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism.
Authors: Qin Yu / Dongchun Ni / Julia Kowal / Ioannis Manolaridis / Scott M Jackson / Henning Stahlberg / Kaspar P Locher /
Abstract: ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the ...ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs.
History
DepositionMay 16, 2021-
Header (metadata) releaseJul 21, 2021-
Map releaseJul 21, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7oji
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12952.png

Downloads & links

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Map

FileDownload / File: emd_12952.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 384 pix.
= 253.44 Å		0.66 Å/pix.
x 384 pix.
= 253.44 Å		0.66 Å/pix.
x 384 pix.
= 253.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.26 / Movie #1: 0.3
Minimum - Maximum-0.6124716 - 1.2754791
Average (Standard dev.)0.0050503924 (±0.04009365)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 253.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.660.660.66
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z253.440253.440253.440
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.6121.2750.005

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Supplemental data

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Mask #1

Fileemd_12952_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12952_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12952_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : ABCG2 in complex with topotecan under turnover condition

EntireName: ABCG2 in complex with topotecan under turnover condition
Components
  • Complex: ABCG2 in complex with topotecan under turnover condition
    • Protein or peptide: Broad substrate specificity ATP-binding cassette transporter ABCG2
  • Ligand: (S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3',4':6,7]INOLIZINO[1,2-B]-QUINOLINE-3,14(4H,12H)-DIONE
  • Ligand: CHOLESTEROL
  • Ligand: DIUNDECYL PHOSPHATIDYL CHOLINE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: ABCG2 in complex with topotecan under turnover condition

SupramoleculeName: ABCG2 in complex with topotecan under turnover condition
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: ABCG2 was incubated with 5mM ATP, 5mM MgCl2, 0.5mM ADP, 100 uM topotecan at room temperature for 10 min
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293EBNA
Molecular weightExperimental: 144 KDa

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Macromolecule #1: Broad substrate specificity ATP-binding cassette transporter ABCG2

MacromoleculeName: Broad substrate specificity ATP-binding cassette transporter ABCG2
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ABC-type xenobiotic transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 73.526938 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDYKDDDDKG SSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAIL GPTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA T TMTNHEKN ...String:
MDYKDDDDKG SSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAIL GPTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA T TMTNHEKN ERINRVIQEL GLDKVADSKV GTQFIRGVSG GERKRTSIGM ELITDPSILF LDEPTTGLDS STANAVLLLL KR MSKQGRT IIFSIHQPRY SIFKLFDSLT LLASGRLMFH GPAQEALGYF ESAGYHCEAY NNPADFFLDI INGDSTAVAL NRE EDFKAT EIIEPSKQDK PLIEKLAEIY VNSSFYKETK AELHQLSGGE KKKKITVFKE ISYTTSFCHQ LRWVSKRSFK NLLG NPQAS IAQIIVTVVL GLVIGAIYFG LKNDSTGIQN RAGVLFFLTT NQCFSSVSAV ELFVVEKKLF IHEYISGYYR VSSYF LGKL LSDLLPMRML PSIIFTCIVY FMLGLKPKAD AFFVMMFTLM MVAYSASSMA LAIAAGQSVV SVATLLMTIC FVFMMI FSG LLVNLTTIAS WLSWLQYFSI PRYGFTALQH NEFLGQNFCP GLNATGNNPC NYATCTGEEY LVKQGIDLSP WGLWKNH VA LACMIVIFLT IAYLKLLFLK KYS

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Macromolecule #2: (S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3'...

MacromoleculeName: (S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3',4':6,7]INOLIZINO[1,2-B]-QUINOLINE-3,14(4H,12H)-DIONE
type: ligand / ID: 2 / Number of copies: 1 / Formula: TTC
Molecular weightTheoretical: 421.446 Da
Chemical component information

ChemComp-TTC:
(S)-10-[(DIMETHYLAMINO)METHYL]-4-ETHYL-4,9-DIHYDROXY-1H-PYRANO[3',4':6,7]INOLIZINO[1,2-B]-QUINOLINE-3,14(4H,12H)-DIONE / medication, inhibitor*YM

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Macromolecule #3: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 4 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #4: DIUNDECYL PHOSPHATIDYL CHOLINE

MacromoleculeName: DIUNDECYL PHOSPHATIDYL CHOLINE / type: ligand / ID: 4 / Number of copies: 2 / Formula: PLC
Molecular weightTheoretical: 622.834 Da
Chemical component information

ChemComp-PLC:
DIUNDECYL PHOSPHATIDYL CHOLINE / phospholipid*YM

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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 mg/mL
BufferpH: 7.5 / Component:
ConcentrationFormulaName
0.15 MNaCl
0.04 MHepes
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Details: 25mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Grids were blotted for 2.5s with blot force 1.
DetailsThe sample was mono-disperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV / Details: no phase plate
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal magnification: 130000
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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Image processing

Particle selectionNumber selected: 2623169
CTF correctionSoftware - Name: cryoSPARC (ver. 3)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 78323
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final 3D classificationNumber classes: 3 / Avg.num./class: 99713 / Software - Name: cryoSPARC (ver. 3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6hco

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-7oji:
ABCG2 topotecan turnover-2 state

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