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- EMDB-6020: Conformational Spectrum of Multidrug ABC Transporters Revealed by... -

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Basic information

Entry
Database: EMDB / ID: EMD-6020
TitleConformational Spectrum of Multidrug ABC Transporters Revealed by Single Particle Electron Microscopy
Map dataP-gp in inward-facing conformation
Sample
  • Sample: Pgp_m stabilised in lipid/amphiphilic environment - inward-facing 6.5 nm opening
  • Protein or peptide: P-glycoprotein
KeywordsABC transporter / P-gp / MsbA
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsMoeller A / Chang Lee S / Tao H / Speir S / Chang G / Urbatsch IL / Potter CS / Carragher B / Zhang Q
CitationJournal: Structure / Year: 2015
Title: Distinct conformational spectrum of homologous multidrug ABC transporters.
Authors: Arne Moeller / Sung Chang Lee / Houchao Tao / Jeffrey A Speir / Geoffrey Chang / Ina L Urbatsch / Clinton S Potter / Bridget Carragher / Qinghai Zhang /
Abstract: ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. ...ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. Significant questions and controversies remain regarding the relevance of their conformations observed in X-ray structures, their structural dynamics, and mechanism of transport. Here, we used single particle electron microscopy (EM) to delineate the entire conformational spectrum of two homologous ABC exporters (bacterial MsbA and mammalian P-glycoprotein) and the influence of nucleotide and substrate binding. Newly developed amphiphiles in complex with lipids that support high protein stability and activity enabled EM visualization of individual complexes in a membrane-mimicking environment. The data provide a comprehensive view of the conformational flexibility of these ABC exporters under various states and demonstrate not only similarities but striking differences between their mechanistic and energetic regulation of conformational changes.
History
DepositionAug 7, 2014-
Header (metadata) releaseSep 24, 2014-
Map releaseFeb 4, 2015-
UpdateSep 16, 2015-
Current statusSep 16, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6020.tif

Downloads & links

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Map

FileDownload / File: emd_6020.map.gz / Format: CCP4 / Size: 245.1 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationP-gp in inward-facing conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.46 Å/pix.
x 40 pix.
= 218.2 Å		5.46 Å/pix.
x 40 pix.
= 218.2 Å		5.46 Å/pix.
x 40 pix.
= 218.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.455 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.67714584 - 2.3875587
Average (Standard dev.)-0.01070552 (±0.28659886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-20-20-20
Dimensions404040
Spacing404040
CellA=B=C: 218.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.4555.4555.455
M x/y/z404040
origin x/y/z0.0000.0000.000
length x/y/z218.200218.200218.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS-20-20-20
NC/NR/NS404040
D min/max/mean-0.6772.388-0.011

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Supplemental data

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Sample components

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Entire : Pgp_m stabilised in lipid/amphiphilic environment - inward-facing...

EntireName: Pgp_m stabilised in lipid/amphiphilic environment - inward-facing 6.5 nm opening
Components
  • Sample: Pgp_m stabilised in lipid/amphiphilic environment - inward-facing 6.5 nm opening
  • Protein or peptide: P-glycoprotein

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Supramolecule #1000: Pgp_m stabilised in lipid/amphiphilic environment - inward-facing...

SupramoleculeName: Pgp_m stabilised in lipid/amphiphilic environment - inward-facing 6.5 nm opening
type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1
Molecular weightTheoretical: 140 KDa

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Macromolecule #1: P-glycoprotein

MacromoleculeName: P-glycoprotein / type: protein_or_peptide / ID: 1 / Name.synonym: P-gp, Pgp, MDR1A / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse / Location in cell: plasma membrane
Molecular weightTheoretical: 140 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus) / Recombinant strain: Gs115 / Recombinant plasmid: pHIL-mdr3.5-His6

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

StainingType: NEGATIVE / Details: uranyl formate
GridDetails: thin carbon over holes
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
DateAug 12, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 456 / Average electron dose: 45 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.75 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsRCT reconstruction
CTF correctionDetails: whole micrograph - unitilted images
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: Appion, Spider, Sparx, Eman2 / Number images used: 145

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