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- EMDB-0190: Cryo-EM structure of the ABCG2 E211Q mutant bound to ATP and Magnesium -

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Basic information

Entry
Database: EMDB / ID: EMD-0190
TitleCryo-EM structure of the ABCG2 E211Q mutant bound to ATP and Magnesium
Map data
Sample
  • Complex: inhibitor-bound ABCG2
    • Complex: A mutant (E211Q) of the ATP-binding cassette sub-family G member 2
      • Protein or peptide: ATP-binding cassette sub-family G member 2
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane / organic anion transmembrane transporter activity / xenobiotic transport across blood-brain barrier / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / Ciprofloxacin ADME / transepithelial transport / cellular detoxification / ABC-type xenobiotic transporter activity / NFE2L2 regulating MDR associated enzymes / Heme biosynthesis / Heme degradation / lipid transport / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / mitochondrial membrane / brush border membrane / Iron uptake and transport / transmembrane transport / apical plasma membrane / membrane raft / ATP hydrolysis activity / protein homodimerization 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) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsManolaridis I / Jackson SM / Taylor NMI / Kowal J / Stahlberg H / Locher KP
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nature / Year: 2018
Title: Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states.
Authors: Ioannis Manolaridis / Scott M Jackson / Nicholas M I Taylor / Julia Kowal / Henning Stahlberg / Kaspar P Locher /
Abstract: ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood- ...ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood-testis and maternal-fetal barriers. Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs. Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies. However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (ES) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of ES can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a 'plug' between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude ES and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.
History
DepositionAug 13, 2018-
Header (metadata) releaseNov 7, 2018-
Map releaseNov 7, 2018-
UpdateOct 23, 2019-
Current statusOct 23, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hbu
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hzm
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0190.png

Downloads & links

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Map

FileDownload / File: emd_0190.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.81 Å
Density
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.27293262 - 0.42899606
Average (Standard dev.)0.0010245538 (±0.010962626)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 207.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.810.810.81
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z207.360207.360207.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2730.4290.001

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Supplemental data

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Sample components

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Entire : inhibitor-bound ABCG2

EntireName: inhibitor-bound ABCG2
Components
  • Complex: inhibitor-bound ABCG2
    • Complex: A mutant (E211Q) of the ATP-binding cassette sub-family G member 2
      • Protein or peptide: ATP-binding cassette sub-family G member 2
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: inhibitor-bound ABCG2

SupramoleculeName: inhibitor-bound ABCG2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1

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Supramolecule #2: A mutant (E211Q) of the ATP-binding cassette sub-family G member 2

SupramoleculeName: A mutant (E211Q) of the ATP-binding cassette sub-family G member 2
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: HOMO SAPIENS (human)
Recombinant expressionOrganism: HOMO SAPIENS (human)

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Macromolecule #1: ATP-binding cassette sub-family G member 2

MacromoleculeName: ATP-binding cassette sub-family G member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 72.384867 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA TTMTNHEKNE R INRVIQEL ...String:
MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA TTMTNHEKNE R INRVIQEL GLDKVADSKV GTQFIRGVSG GERKRTSIGM ELITDPSILF LDQPTTGLDS STANAVLLLL KRMSKQGRTI IF SIHQPRY SIFKLFDSLT LLASGRLMFH GPAQEALGYF ESAGYHCEAY NNPADFFLDI INGDSTAVAL NREEDFKATE IIE PSKQDK PLIEKLAEIY VNSSFYKETK AELHQLSGGE KKKKITVFKE ISYTTSFCHQ LRWVSKRSFK NLLGNPQASI AQII VTVVL GLVIGAIYFG LKNDSTGIQN RAGVLFFLTT NQCFSSVSAV ELFVVEKKLF IHEYISGYYR VSSYFLGKLL SDLLP MRML PSIIFTCIVY FMLGLKPKAD AFFVMMFTLM MVAYSASSMA LAIAAGQSVV SVATLLMTIC FVFMMIFSGL LVNLTT IAS WLSWLQYFSI PRYGFTALQH NEFLGQNFCP GLNATGNNPC NYATCTGEEY LVKQGIDLSP WGLWKNHVAL ACMIVIF LT IAYLKLLFLK KYS

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

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

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

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.4 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 4905 / Average exposure time: 0.2 sec. / Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY / Details: 6ETI
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 288447
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementOverall B value: 136
Output model

PDB-6hbu:
Cryo-EM structure of the ABCG2 E211Q mutant bound to ATP and Magnesium

PDB-6hzm:
Cryo-EM structure of the ABCG2 E211Q mutant bound to ATP and Magnesium (alternative placement of Magnesium into the cryo-EM density)

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