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Yorodumi- EMDB-0196: Cryo-EM structure of the ABCG2 E211Q mutant bound to estrone 3-su... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0196 | |||||||||
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Title | Cryo-EM structure of the ABCG2 E211Q mutant bound to estrone 3-sulfate and 5D3-Fab | |||||||||
Map data | ABCG2 in complex with estrone-3-sulfate and 5D3-Fab | |||||||||
Sample |
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Function / homology | Function and homology information biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / sphingolipid transporter activity / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / sphingolipid transporter activity / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / 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 / cellular detoxification / Heme biosynthesis / Heme degradation / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / mitochondrial membrane / brush border membrane / Iron uptake and transport / transmembrane transport / membrane raft / apical plasma membrane / protein homodimerization activity / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / plasma membrane Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / Mus musculus (house mouse) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.58 Å | |||||||||
Authors | Manolaridis I / Jackson SM / Taylor NMI / Kowal J / Stahlberg H / Locher KP | |||||||||
Funding support | Switzerland, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_0196.map.gz | 197.8 MB | EMDB map data format | |
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Header (meta data) | emd-0196-v30.xml emd-0196.xml | 15 KB 15 KB | Display Display | EMDB header |
Images | emd_0196.png | 61.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0196 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0196 | HTTPS FTP |
-Validation report
Summary document | emd_0196_validation.pdf.gz | 264.3 KB | Display | EMDB validaton report |
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Full document | emd_0196_full_validation.pdf.gz | 263.4 KB | Display | |
Data in XML | emd_0196_validation.xml.gz | 7.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0196 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0196 | HTTPS FTP |
-Related structure data
Related structure data | 6hcoMC 0190C 6hbuC 6hzmC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_0196.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | ABCG2 in complex with estrone-3-sulfate and 5D3-Fab | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.812 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : nanodisc-reconsituted ABCG2 E211Q mutant bound to estrone 3-sulfa...
Entire | Name: nanodisc-reconsituted ABCG2 E211Q mutant bound to estrone 3-sulfate and 5D3-Fab |
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Components |
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-Supramolecule #1: nanodisc-reconsituted ABCG2 E211Q mutant bound to estrone 3-sulfa...
Supramolecule | Name: nanodisc-reconsituted ABCG2 E211Q mutant bound to estrone 3-sulfate and 5D3-Fab type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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-Supramolecule #2: ABCG2 E211Q mutant
Supramolecule | Name: ABCG2 E211Q mutant / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293 EBNA |
-Supramolecule #3: 5D3-Fab
Supramolecule | Name: 5D3-Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Mus musculus (house mouse) |
-Macromolecule #1: ATP-binding cassette sub-family G member 2
Macromolecule | Name: ATP-binding cassette sub-family G member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 73.394758 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: DYKDDDDKGS SSSNVEVFIP VSQGNTNGFP ATASNDLKAF TEGAVLSFHN ICYRVKLKSG FLPCRKPVEK EILSNINGIM KPGLNAILG PTGGGKSSLL DVLAARKDPS GLSGDVLING APRPANFKCN SGYVVQDDVV MGTLTVRENL QFSAALRLAT T MTNHEKNE ...String: DYKDDDDKGS SSSNVEVFIP VSQGNTNGFP ATASNDLKAF TEGAVLSFHN ICYRVKLKSG FLPCRKPVEK EILSNINGIM KPGLNAILG PTGGGKSSLL DVLAARKDPS GLSGDVLING APRPANFKCN SGYVVQDDVV MGTLTVRENL QFSAALRLAT T MTNHEKNE RINRVIQELG LDKVADSKVG TQFIRGVSGG ERKRTSIGME LITDPSILFL DQPTTGLDSS TANAVLLLLK RM SKQGRTI IFSIHQPRYS IFKLFDSLTL LASGRLMFHG PAQEALGYFE SAGYHCEAYN NPADFFLDII NGDSTAVALN REE DFKATE IIEPSKQDKP LIEKLAEIYV NSSFYKETKA ELHQLSGGEK KKKITVFKEI SYTTSFCHQL RWVSKRSFKN LLGN PQASI AQIIVTVVLG LVIGAIYFGL KNDSTGIQNR AGVLFFLTTN QCFSSVSAVE LFVVEKKLFI HEYISGYYRV SSYFL GKLL SDLLPMRMLP SIIFTCIVYF MLGLKPKADA FFVMMFTLMM VAYSASSMAL AIAAGQSVVS VATLLMTICF VFMMIF SGL LVNLTTIASW LSWLQYFSIP RYGFTALQHN EFLGQNFCPG LNATGNNPCN YATCTGEEYL VKQGIDLSPW GLWKNHV AL ACMIVIFLTI AYLKLLFLKK YS |
-Macromolecule #2: 5D3-Fab light chain
Macromolecule | Name: 5D3-Fab light chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 23.594016 KDa |
Recombinant expression | Organism: Mus musculus (house mouse) |
Sequence | String: DIVLTQSPSS FSVSLGDRVT ISCKASGYIL NRLAWYQQKP GNAPRLLISG ATSLETGFPS RFSGTGSGKD YTLSISSLQT EDVGTYYCQ QYWSTPWTFG GGTKLEIRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS ...String: DIVLTQSPSS FSVSLGDRVT ISCKASGYIL NRLAWYQQKP GNAPRLLISG ATSLETGFPS RFSGTGSGKD YTLSISSLQT EDVGTYYCQ QYWSTPWTFG GGTKLEIRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRNEC |
-Macromolecule #3: 5D3-Fab heavy chain
Macromolecule | Name: 5D3-Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 23.843633 KDa |
Recombinant expression | Organism: Mus musculus (house mouse) |
Sequence | String: QVQLQESGPG LVKPSQSLSL TCTVTGFSIT SDYAWNWIRQ FPGKKLEWMG YINFDGGTTY NPSLRGRISI TRDTSKNQFF LQLRSVTPE DTATYYCATF YGAKGTLDYW GQGTSVTVSS AKTTPPSVYP LAPVCGDTSG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG ...String: QVQLQESGPG LVKPSQSLSL TCTVTGFSIT SDYAWNWIRQ FPGKKLEWMG YINFDGGTTY NPSLRGRISI TRDTSKNQFF LQLRSVTPE DTATYYCATF YGAKGTLDYW GQGTSVTVSS AKTTPPSVYP LAPVCGDTSG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VTSSTWPSQS ITCNVAHPAS STKVDKKIEP RGP |
-Macromolecule #5: estrone 3-sulfate
Macromolecule | Name: estrone 3-sulfate / type: ligand / ID: 5 / Number of copies: 1 / Formula: FY5 |
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Molecular weight | Theoretical: 350.429 Da |
Chemical component information | ChemComp-FY5: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.4 mg/mL |
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Buffer | pH: 7.5 |
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 3984 / Average exposure time: 0.2 sec. / Average electron dose: 100.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 42790 |
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Initial angle assignment | Type: PROJECTION MATCHING |
Final angle assignment | Type: PROJECTION MATCHING |