+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2715 | |||||||||
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Title | Electron cryo-microscopy of ABCG2 from two-dimensional crystals | |||||||||
Map data | Non-crystallographic symmetry averaging around the local C2 symmetry axis was applied to the raw ABCG2 map. | |||||||||
Sample |
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Keywords | ABCG2 / BCRP / Cryo-EM / 3D structure / ABC transporter | |||||||||
Function / homology | Function and homology information transport / small molecule metabolic process / biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / heme transport / renal urate salt excretion / sphingolipid transporter activity / urate metabolic process ...transport / small molecule metabolic process / biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / heme transport / renal urate salt excretion / sphingolipid transporter activity / urate metabolic process / urate transmembrane transporter activity / response to xenobiotic stimulus => GO:0009410 / 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 transmembrane transport / xenobiotic transport across blood-brain barrier / transepithelial transport / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / Ciprofloxacin ADME / xenobiotic transport / 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 / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / brush border membrane / Iron uptake and transport / mitochondrial membrane / transmembrane transport / intracellular iron ion homeostasis / apical plasma membrane / membrane raft / protein homodimerization activity / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / plasma membrane Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | electron crystallography / cryo EM / negative staining / Resolution: 18.0 Å | |||||||||
Authors | Rosenberg MF / Bikadi Z / Hazai E / Starborg T / Kelley L / Chayen NE / Ford RC / Mao Q | |||||||||
Citation | Journal: Acta Crystallogr D Biol Crystallogr / Year: 2015 Title: Three-dimensional structure of the human breast cancer resistance protein (BCRP/ABCG2) in an inward-facing conformation. Authors: Mark F Rosenberg / Zsolt Bikadi / Eszter Hazai / Tobias Starborg / Lawrence Kelley / Naomi E Chayen / Robert C Ford / Qingcheng Mao / Abstract: ABCG2 is an efflux drug transporter that plays an important role in drug resistance and drug disposition. In this study, the first three-dimensional structure of human full-length ABCG2 analysed by ...ABCG2 is an efflux drug transporter that plays an important role in drug resistance and drug disposition. In this study, the first three-dimensional structure of human full-length ABCG2 analysed by electron crystallography from two-dimensional crystals in the absence of nucleotides and transported substrates is reported at 2 nm resolution. In this state, ABCG2 forms a symmetric homodimer with a noncrystallographic twofold axis perpendicular to the two-dimensional crystal plane, as confirmed by subtomogram averaging. This configuration suggests an inward-facing configuration similar to murine ABCB1, with the nucleotide-binding domains (NBDs) widely separated from each other. In the three-dimensional map, densities representing the long cytoplasmic extensions from the transmembrane domains that connect the NBDs are clearly visible. The structural data have allowed the atomic model of ABCG2 to be refined, in which the two arms of the V-shaped ABCG2 homodimeric complex are in a more closed and narrower conformation. The structural data and the refined model of ABCG2 are compatible with the biochemical analysis of the previously published mutagenesis studies, providing novel insight into the structure and function of the transporter. | |||||||||
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_2715.map.gz | 78.5 KB | EMDB map data format | |
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Header (meta data) | emd-2715-v30.xml emd-2715.xml | 13.3 KB 13.3 KB | Display Display | EMDB header |
Images | EMD-2715-abcg2_picture24714.tiff | 952.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2715 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2715 | HTTPS FTP |
-Validation report
Summary document | emd_2715_validation.pdf.gz | 166.2 KB | Display | EMDB validaton report |
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Full document | emd_2715_full_validation.pdf.gz | 165.3 KB | Display | |
Data in XML | emd_2715_validation.xml.gz | 4.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2715 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2715 | HTTPS FTP |
-Related structure data
Similar structure data | Similarity search - Function & homologyF&H Search |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2715.map.gz / Format: CCP4 / Size: 2.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Non-crystallographic symmetry averaging around the local C2 symmetry axis was applied to the raw ABCG2 map. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X: 3.2 Å / Y: 3.2 Å / Z: 3.3 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 : ABCG2 (breast cancer resistance protein).
Entire | Name: ABCG2 (breast cancer resistance protein). |
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Components |
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-Supramolecule #1000: ABCG2 (breast cancer resistance protein).
Supramolecule | Name: ABCG2 (breast cancer resistance protein). / type: sample / ID: 1000 / Details: Two-dimensional crystals of purified ABCG2. / Oligomeric state: Homodimer / Number unique components: 1 |
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Molecular weight | Experimental: 70 KDa / Theoretical: 70 KDa / Method: SDS-PAGE. |
-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 / Name.synonym: Breast cancer resistance protein Details: Purified ABCG2 formed two-dimensional crystals on a carbon electron microscopy grid. Number of copies: 2 / Oligomeric state: Homodimer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma membrane |
Molecular weight | Experimental: 70 KDa / Theoretical: 70 KDa |
Recombinant expression | Organism: Komagataella pastoris (fungus) / Recombinant strain: KM71 / Recombinant plasmid: pHIL-BCRP-His10 |
Sequence | UniProtKB: Broad substrate specificity ATP-binding cassette transporter ABCG2 GO: intracellular iron ion homeostasis, xenobiotic transmembrane transport, heme transport, response to xenobiotic stimulus => GO:0009410, small molecule metabolic process, transmembrane transport, ...GO: intracellular iron ion homeostasis, xenobiotic transmembrane transport, heme transport, response to xenobiotic stimulus => GO:0009410, small molecule metabolic process, transmembrane transport, transport, urate metabolic process, xenobiotic transport InterPro: AAA+ ATPase domain, ABC-2 type transporter, ABC transporter-like, ATP-binding domain, P-loop containing nucleoside triphosphate hydrolase |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | electron crystallography |
Aggregation state | 2D array |
-Sample preparation
Concentration | 0.3 mg/mL |
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Buffer | pH: 8 Details: 50 mM Tris-HCl , 10% glycerol, 1 mM 2-mercaptoethanol, and 0.187 mg/ml DDM. |
Staining | Type: NEGATIVE Details: Some grids were negatively stained with 2 % w/v uranyl-acetate for 60 seconds. |
Grid | Details: 300 mesh gold/carbon grids with thick carbon support (5-10 nm) . |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 98 K / Instrument: FEI VITROBOT MARK III Method: 2-D crystals growing epitaxially on the grid surface were blotted for 1 second before plunging. |
Details | Crystals were grown epitaxially on the surface of carbon grids. |
Crystal formation | Details: Crystals were grown epitaxially on the surface of carbon grids. |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Temperature | Min: 80 K / Max: 105 K / Average: 93 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 130,000 times magnification. |
Date | Oct 1, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 76 / Average electron dose: 12 e/Å2 / Bits/pixel: 16 |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 79730 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.02 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder: Liquid nitrogen cooled. Standard Polara cartridge. Specimen holder model: OTHER / Tilt angle max: 60 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 60 ° |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
Details | Images were unbent using 2dx. |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: 2dx / Details: Final maps were calculated with CCP4. |
Crystal parameters | Unit cell - A: 70 Å / Unit cell - B: 123 Å / Unit cell - C: 200 Å / Unit cell - γ: 90 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 1 21 |
CTF correction | Details: Algorithm in 2dx. |