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- EMDB-5525: Electron cryo-microscopy of ABC BmrA in 12-fold symmetry rings -

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Basic information

Entry
Database: EMDB / ID: EMD-5525
TitleElectron cryo-microscopy of ABC BmrA in 12-fold symmetry rings
Map dataReconstruction of BmrA in a lipidic environment. Reconstruction is D12 symmetrized. 24 homodimers of BmrA are inserted into a lipid bilayer.
Sample
  • Sample: BmrA reconstituted at high density into lipid bilayer.
  • Protein or peptide: BmrA
KeywordsABC transporter / exporter / bmra / yvcc / transmembrane protein / reconstitution into lipids / membrane protein
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 23.0 Å
AuthorsFribourg PF / Chami M / Sorzano CO / Gubellini F / Marabini R / Marco S / Jault JM / Levy D
CitationJournal: J Mol Biol / Year: 2014
Title: 3D cryo-electron reconstruction of BmrA, a bacterial multidrug ABC transporter in an inward-facing conformation and in a lipidic environment.
Authors: Pierre Frederic Fribourg / Mohamed Chami / Carlos Oscar S Sorzano / Francesca Gubellini / Roberto Marabini / Sergio Marco / Jean-Michel Jault / Daniel Lévy /
Abstract: ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether ...ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3 nm and 2.5 nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3 nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6 nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane.
History
DepositionNov 2, 2012-
Header (metadata) releaseNov 21, 2012-
Map releaseNov 21, 2012-
UpdateMay 14, 2014-
Current statusMay 14, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.029
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.029
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5525.tif

Downloads & links

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Map

FileDownload / File: emd_5525.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of BmrA in a lipidic environment. Reconstruction is D12 symmetrized. 24 homodimers of BmrA are inserted into a lipid bilayer.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.5 Å/pix.
x 200 pix.
= 500. Å		2.5 Å/pix.
x 200 pix.
= 500. Å		2.5 Å/pix.
x 200 pix.
= 500. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.029 / Movie #1: 0.029
Minimum - Maximum-0.03243274 - 0.06041811
Average (Standard dev.)0.00049039 (±0.01073864)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 500.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z500.000500.000500.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0320.0600.000

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Supplemental data

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Sample components

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Entire : BmrA reconstituted at high density into lipid bilayer.

EntireName: BmrA reconstituted at high density into lipid bilayer.
Components
  • Sample: BmrA reconstituted at high density into lipid bilayer.
  • Protein or peptide: BmrA

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Supramolecule #1000: BmrA reconstituted at high density into lipid bilayer.

SupramoleculeName: BmrA reconstituted at high density into lipid bilayer.
type: sample / ID: 1000
Oligomeric state: 24 Homodimers of BmrA inserted into lipid bilayer
Number unique components: 1
Molecular weightExperimental: 3.12 MDa / Theoretical: 3.12 MDa

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Macromolecule #1: BmrA

MacromoleculeName: BmrA / type: protein_or_peptide / ID: 1 / Name.synonym: YvcC / Details: 24 homodimers of inserted into lipid bilayer / Number of copies: 24 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightExperimental: 3.12 MDa / Theoretical: 3.12 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.1 mg/mL
BufferpH: 7.8 / Details: 50mM HEPES, 100mM NaCl
GridDetails: Ted Pella inc. holey formvar grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: specimen linked to a Ni++-NTA-DOGS/DOPC lipid monolayer
Method: Blot for 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
DateJan 1, 2003
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON COOLSCAN / Number real images: 102 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.6 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsXmipp CL2D classification and Projection matching refinement with Invert Fourier 3D reconstruction
CTF correctionDetails: Micrograph based
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp / Number images used: 1188

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Atomic model buiding 1

Initial modelPDB ID:

3g61

SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross correlation factor

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