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- EMDB-6085: Single particle cryo-EM reconstruction of ABC transporter TmrAB i... -

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Basic information

Entry
Database: EMDB / ID: EMD-6085
TitleSingle particle cryo-EM reconstruction of ABC transporter TmrAB in complex with a Fab AH5
Map data3D reconstruction of TmrAB in complex with Fab AH5
Sample
  • Sample: TmrAB in complex with Fab AH5
  • Protein or peptide: ABC exporter
  • Protein or peptide: Fab AH5
KeywordsABC transporter / TmrAB
Function / homology
Function and homology information


ABC-type transporter activity / ATP hydrolysis activity / ATP binding / membrane
Similarity search - Function
Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Multidrug resistance ABC transporter ATP-binding and permease protein
Similarity search - Component
Biological speciesThermus thermophilus (bacteria) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsKim JM / Wu S / Tomasiak T / Mergel C / Winter MB / Stiller S / Robles-Colmanares Y / Stroud RM / Tampe R / Craik CS / Cheng Y
CitationJournal: Nature / Year: 2015
Title: Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter.
Authors: JungMin Kim / Shenping Wu / Thomas M Tomasiak / Claudia Mergel / Michael B Winter / Sebastian B Stiller / Yaneth Robles-Colmanares / Robert M Stroud / Robert Tampé / Charles S Craik / Yifan Cheng /
Abstract: ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains. ABC exporters are ...ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains. ABC exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases. TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus; it is homologous to various multidrug transporters and contains one degenerate site with a non-catalytic residue next to the Walker B motif. Here we report a subnanometre-resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single-particle electron cryomicroscopy. The reconstructions clearly resolve characteristic features of ABC transporters, including helices in the transmembrane domain and nucleotide-binding domains. A cavity in the transmembrane domain is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two nucleotide-binding domains remain in contact via their carboxy-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two nucleotide-binding domains during the transition from the inward-facing to outward-facing conformations.
History
DepositionSep 7, 2014-
Header (metadata) releaseOct 8, 2014-
Map releaseNov 5, 2014-
UpdateJan 21, 2015-
Current statusJan 21, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6085.gif

Downloads & links

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Map

FileDownload / File: emd_6085.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of TmrAB in complex with Fab AH5
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.96 Å/pix.
x 128 pix.
= 250.88 Å		1.96 Å/pix.
x 128 pix.
= 250.88 Å		1.96 Å/pix.
x 128 pix.
= 250.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0 / Movie #1: 4
Minimum - Maximum-15.689664840000001 - 27.532846450000001
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 250.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.961.961.96
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z250.880250.880250.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-15.69027.5330.000

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Supplemental data

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Sample components

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Entire : TmrAB in complex with Fab AH5

EntireName: TmrAB in complex with Fab AH5
Components
  • Sample: TmrAB in complex with Fab AH5
  • Protein or peptide: ABC exporter
  • Protein or peptide: Fab AH5

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Supramolecule #1000: TmrAB in complex with Fab AH5

SupramoleculeName: TmrAB in complex with Fab AH5 / type: sample / ID: 1000 / Details: single particle; sample was monodisperse / Oligomeric state: One heterodimer with one Fab bound / Number unique components: 2
Molecular weightExperimental: 185 KDa / Theoretical: 185 KDa

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Macromolecule #1: ABC exporter

MacromoleculeName: ABC exporter / type: protein_or_peptide / ID: 1 / Name.synonym: TmrAB / Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: Yes
Source (natural)Organism: Thermus thermophilus (bacteria) / synonym: thermophilic Gram-negative eubacterium
Molecular weightExperimental: 185 KDa / Theoretical: 185 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Multidrug resistance ABC transporter ATP-binding and permease protein

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Macromolecule #2: Fab AH5

MacromoleculeName: Fab AH5 / type: protein_or_peptide / ID: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

GridDetails: Quantifoil grid
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI POLARA 300
DetailsDose fractionation and motion correction
DateJan 1, 2012
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 40 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 20000
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 102000

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