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- EMDB-2714: Negative stain reconstruction of AcrB/SMALP complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2714
TitleNegative stain reconstruction of AcrB/SMALP complex
Map dataReconstruction of AcrB encapsulated in a SMALP polymer
Sample
  • Sample: E. coli AcrB in a SMALP scaffold
  • Protein or peptide: Acridine resistance protein B
KeywordsAcrB / negative stain / SMALP
Function / homology
Function and homology information


alkane transmembrane transporter activity / alkane transport / enterobactin transport / enterobactin transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the cell outer membrane / periplasmic side of plasma membrane / efflux pump complex / bile acid transmembrane transporter activity / xenobiotic transport / bile acid and bile salt transport ...alkane transmembrane transporter activity / alkane transport / enterobactin transport / enterobactin transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the cell outer membrane / periplasmic side of plasma membrane / efflux pump complex / bile acid transmembrane transporter activity / xenobiotic transport / bile acid and bile salt transport / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / fatty acid transport / response to toxic substance / response to xenobiotic stimulus / response to antibiotic / identical protein binding / membrane / plasma membrane
Similarity search - Function
Hydrophobe/amphiphile efflux-1 HAE1 / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / Acriflavin resistance protein / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Multidrug efflux pump subunit AcrB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsPostis V / Rawson S / Mitchell JK / Lee SC / Parslow R / Dafforn TR / Baldwin SA / Muench SP
CitationJournal: Biochim Biophys Acta / Year: 2015
Title: The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy.
Authors: Vincent Postis / Shaun Rawson / Jennifer K Mitchell / Sarah C Lee / Rosemary A Parslow / Tim R Dafforn / Stephen A Baldwin / Stephen P Muench /
Abstract: Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances ...Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving >3.5Å resolution detail in membrane proteins of modest (~300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function.
History
DepositionJul 18, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseDec 24, 2014-
UpdateJan 7, 2015-
Current statusJan 7, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2714.png

Downloads & links

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Map

FileDownload / File: emd_2714.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of AcrB encapsulated in a SMALP polymer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4 Å/pix.
x 128 pix.
= 512. Å		4 Å/pix.
x 128 pix.
= 512. Å		4 Å/pix.
x 128 pix.
= 512. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-7.28567362 - 9.009478570000001
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 512.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z444
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z512.000512.000512.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ442626
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-7.2869.009-0.000

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Supplemental data

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Sample components

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Entire : E. coli AcrB in a SMALP scaffold

EntireName: E. coli AcrB in a SMALP scaffold
Components
  • Sample: E. coli AcrB in a SMALP scaffold
  • Protein or peptide: Acridine resistance protein B

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Supramolecule #1000: E. coli AcrB in a SMALP scaffold

SupramoleculeName: E. coli AcrB in a SMALP scaffold / type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: Trimeric AcrB / Number unique components: 1
Molecular weightExperimental: 360 KDa / Theoretical: 360 KDa

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Macromolecule #1: Acridine resistance protein B

MacromoleculeName: Acridine resistance protein B / type: protein_or_peptide / ID: 1 / Name.synonym: AcrB / Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: E. coli / Location in cell: membrane
Molecular weightExperimental: 360 KDa / Theoretical: 360 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Multidrug efflux pump subunit AcrB

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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

Concentration0.12 mg/mL
BufferpH: 8
Details: 50mM Tris-HCl, pH 8.0, 10%(v/v) glycerol and 500mM NaCl
StainingType: NEGATIVE / Details: 1% Uranyl acetate
GridDetails: Agar Carbon support film 300 mesh
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 80,000 times magnification
DetailsLow Dose
DateFeb 6, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ORIUS SC200 (2k x 2k) / Number real images: 307 / Average electron dose: 50 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 37500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: OTHER

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Image processing

DetailsParticles were picked manually using EMAN2 boxer program
CTF correctionDetails: Each micrograph CTF-find 3
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: RELION
Details: Gold standard FSC used in RELION for resolution determination. A simple elipsoid used as a starting model
Number images used: 6884

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Atomic model buiding 1

Initial modelPDB ID:

1iwg


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C
SoftwareName: Chmiera
DetailsThe crystal structure was fitted as a rigid body trimer
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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