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- EMDB-22050: 3.9 Angstrom reconstruction of E.coli AcrB embedded in the liposome -

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Basic information

Entry
Database: EMDB / ID: EMD-22050
Title3.9 Angstrom reconstruction of E.coli AcrB embedded in the liposome
Map data3D reconstruction of AcrB after Density Modification
Sample
  • Complex: E.coli AcrB
Function / homology
Function and homology information


xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / periplasmic side of plasma membrane / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / outer membrane-bounded periplasmic space / membrane / identical protein binding / plasma membrane
Similarity search - Function
Hydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Multidrug efflux pump subunit AcrB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsXia Y / Fan X / Yan N
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Cryo-EM analysis of a membrane protein embedded in the liposome.
Authors: Xia Yao / Xiao Fan / Nieng Yan /
Abstract: Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron ...Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.
History
DepositionMay 25, 2020-
Header (metadata) releaseJul 15, 2020-
Map releaseJul 15, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.36
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.36
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22050.png

Downloads & links

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Map

FileDownload / File: emd_22050.map.gz / Format: CCP4 / Size: 7.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of AcrB after Density Modification
Voxel sizeX=Y=Z: 1.114 Å
Density
Contour LevelBy AUTHOR: 0.36 / Movie #1: 0.36
Minimum - Maximum-1.6803911 - 2.4126687
Average (Standard dev.)-0.0000000000 (±0.17928194)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin433343
Dimensions118137116
Spacing116118137
CellA: 129.224 Å / B: 131.452 Å / C: 152.618 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1141.1141.114
M x/y/z116118137
origin x/y/z0.0000.0000.000
length x/y/z129.224131.452152.618
α/β/γ90.00090.00090.000
start NX/NY/NZ434333
NX/NY/NZ116118137
MAP C/R/S321
start NC/NR/NS334343
NC/NR/NS137118116
D min/max/mean-1.6802.413-0.000

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Supplemental data

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Additional map: 3D reconstruction of AcrB after postprocess

Fileemd_22050_additional_1.map
Annotation3D reconstruction of AcrB after postprocess
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered 3D reconstruction of AcrB

Fileemd_22050_additional_2.map
AnnotationUnfiltered 3D reconstruction of AcrB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E.coli AcrB

EntireName: E.coli AcrB
Components
  • Complex: E.coli AcrB

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Supramolecule #1: E.coli AcrB

SupramoleculeName: E.coli AcrB / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 350 kDa/nm

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS / Details: Ozonated
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal magnification: 105000
Specialist opticsSpherical aberration corrector: Cs corrector applied / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 4.0 µm / Digitization - Frames/image: 1-32 / Number grids imaged: 1 / Number real images: 5757 / Average exposure time: 5.6 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware: (Name: CTFFIND (ver. 4.15), RELION (ver. 3.1))
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 65317
FSC plot (resolution estimation)

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