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- EMDB-2715: Electron cryo-microscopy of ABCG2 from two-dimensional crystals -

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Basic information

Entry
Database: EMDB / ID: EMD-2715
TitleElectron cryo-microscopy of ABCG2 from two-dimensional crystals
Map dataNon-crystallographic symmetry averaging around the local C2 symmetry axis was applied to the raw ABCG2 map.
Sample
  • Sample: ABCG2 (breast cancer resistance protein).
  • Protein or peptide: ATP-binding cassette sub-family G member 2
KeywordsABCG2 / BCRP / Cryo-EM / 3D structure / ABC transporter
Function / homology
Function and homology information


response to xenobiotic stimulus => GO:0009410 / transport / small molecule metabolic process / biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / heme transport / sphingolipid transporter activity / renal urate salt excretion ...response to xenobiotic stimulus => GO:0009410 / transport / small molecule metabolic process / biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / heme transport / sphingolipid transporter activity / renal urate salt excretion / Abacavir transmembrane transport / urate metabolic process / urate transmembrane transporter activity / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / external side of apical plasma membrane / xenobiotic transport across blood-brain barrier / xenobiotic transmembrane transport / organic anion transport / : / transepithelial transport / Ciprofloxacin ADME / export across plasma membrane / Paracetamol ADME / NFE2L2 regulating MDR associated enzymes / Differentiation of Keratinocytes in Interfollicular Epidermis in Mammalian Skin / ABC-type xenobiotic transporter / Heme biosynthesis / cellular detoxification / ABC-type xenobiotic transporter activity / Heme degradation / xenobiotic transport / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / transport across blood-brain barrier / 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
ABC transporter family G domain / ABC-2 type transporter / : / ABC-2 type transporter / ABC-2 type transporter / ABC-2 type transporter / ABC transporter / ABC transporter-like, ATP-binding domain / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. ...ABC transporter family G domain / ABC-2 type transporter / : / ABC-2 type transporter / ABC-2 type transporter / ABC-2 type transporter / ABC transporter / ABC transporter-like, ATP-binding domain / 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 / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / 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)
Methodelectron crystallography / cryo EM / negative staining / Resolution: 18.0 Å
AuthorsRosenberg MF / Bikadi Z / Hazai E / Starborg T / Kelley L / Chayen NE / Ford RC / Mao Q
CitationJournal: 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
DepositionJul 23, 2014-
Header (metadata) releaseAug 6, 2014-
Map releaseJul 15, 2015-
UpdateAug 19, 2015-
Current statusAug 19, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.8
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2715-abc...

Downloads & links

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Map

FileDownload / File: emd_2715.map.gz / Format: CCP4 / Size: 2.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNon-crystallographic symmetry averaging around the local C2 symmetry axis was applied to the raw ABCG2 map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.3 Å/pix.
x 88 pix.
= 290.4 Å		3.2 Å/pix.
x 72 pix.
= 230.4 Å		3.2 Å/pix.
x 117 pix.
= 374.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 3.2 Å / Y: 3.2 Å / Z: 3.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.8 / Movie #1: 1.8
Minimum - Maximum-7.10321522 - 13.60273933
Average (Standard dev.)0.01877212 (±0.38814983)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-37-4717
Dimensions7211788
Spacing7211788
CellA: 374.4 Å / B: 230.40001 Å / C: 290.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.23.23.3
M x/y/z1177288
origin x/y/z0.0000.0000.000
length x/y/z374.400230.400290.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-47-3717
NC/NR/NS1177288
D min/max/mean-7.10313.6030.019

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Supplemental data

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Sample components

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Entire : ABCG2 (breast cancer resistance protein).

EntireName: ABCG2 (breast cancer resistance protein).
Components
  • Sample: ABCG2 (breast cancer resistance protein).
  • Protein or peptide: ATP-binding cassette sub-family G member 2

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Supramolecule #1000: ABCG2 (breast cancer resistance protein).

SupramoleculeName: ABCG2 (breast cancer resistance protein). / type: sample / ID: 1000 / Details: Two-dimensional crystals of purified ABCG2. / Oligomeric state: Homodimer / Number unique components: 1
Molecular weightExperimental: 70 KDa / Theoretical: 70 KDa / Method: SDS-PAGE.

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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 / 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma membrane
Molecular weightExperimental: 70 KDa / Theoretical: 70 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus) / Recombinant strain: KM71 / Recombinant plasmid: pHIL-BCRP-His10
SequenceUniProtKB: 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

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 8
Details: 50 mM Tris-HCl , 10% glycerol, 1 mM 2-mercaptoethanol, and 0.187 mg/ml DDM.
StainingType: NEGATIVE
Details: Some grids were negatively stained with 2 % w/v uranyl-acetate for 60 seconds.
GridDetails: 300 mesh gold/carbon grids with thick carbon support (5-10 nm) .
VitrificationCryogen 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.
DetailsCrystals were grown epitaxially on the surface of carbon grids.
Crystal formationDetails: Crystals were grown epitaxially on the surface of carbon grids.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 105 K / Average: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 130,000 times magnification.
DateOct 1, 2013
Image recordingCategory: 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 min0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen 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

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Image processing

DetailsImages were unbent using 2dx.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: 2dx / Details: Final maps were calculated with CCP4.
Crystal parametersUnit 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 correctionDetails: Algorithm in 2dx.

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