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- EMDB-6025: Conformational Spectrum of Multidrug ABC Transporters Revealed by... -

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Basic information

Entry
Database: EMDB / ID: EMD-6025
TitleConformational Spectrum of Multidrug ABC Transporters Revealed by Single Particle Electron Microscopy
Map dataMsbA in inward-facing conformation
Sample
  • Sample: MsbA stabilised in amphiphilic environment - inward-facing 4.5 nm opening
  • Protein or peptide: MsbA
KeywordsABC transporter / P-gp / MsbA
Biological speciesEscherichia coli (E. coli)
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: 1.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6025.tif

Downloads & links

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Map

FileDownload / File: emd_6025.map.gz / Format: CCP4 / Size: 232.4 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMsbA in inward-facing conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5 Å/pix.
x 42 pix.
= 210. Å		5 Å/pix.
x 40 pix.
= 200. Å		5 Å/pix.
x 36 pix.
= 180. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-1.53283215 - 4.16564178
Average (Standard dev.)-0.00299926 (±0.42446631)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-38-37-40
Dimensions403642
Spacing403642
CellA: 180.0 Å / B: 200.0 Å / C: 210.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z555
M x/y/z364042
origin x/y/z0.0000.0000.000
length x/y/z180.000200.000210.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS-37-38-40
NC/NR/NS364042
D min/max/mean-1.5334.166-0.003

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Supplemental data

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Sample components

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Entire : MsbA stabilised in amphiphilic environment - inward-facing 4.5 nm...

EntireName: MsbA stabilised in amphiphilic environment - inward-facing 4.5 nm opening
Components
  • Sample: MsbA stabilised in amphiphilic environment - inward-facing 4.5 nm opening
  • Protein or peptide: MsbA

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Supramolecule #1000: MsbA stabilised in amphiphilic environment - inward-facing 4.5 nm...

SupramoleculeName: MsbA stabilised in amphiphilic environment - inward-facing 4.5 nm opening
type: sample / ID: 1000 / Oligomeric state: homodimer / Number unique components: 1
Molecular weightTheoretical: 140 KDa

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Macromolecule #1: MsbA

MacromoleculeName: MsbA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: plasma membrane
Molecular weightTheoretical: 140 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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
DateJan 24, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 558 / 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: 148

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