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- EMDB-3924: Stalled E. coli ribosomes (Fo-c SecM nascent chains) and native E... -

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Basic information

Entry
Database: EMDB / ID: 3924
TitleStalled E. coli ribosomes (Fo-c SecM nascent chains) and native E. coli membranes containing recombinant YidC
Map dataAverage of subvolumes in class 6
SampleRibosome-nascent chains with native E.coli membranes containing YidC
  • E. coli native membranes containing YidC
  • Ribosomes with SecM stalled nascent chains of ATP synthase subunit c
SourceEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / 100 Å resolution
AuthorsBaker LA / Gruenewald K
CitationJournal: Structure / Year: 2018
Title: Combined H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments.
Authors: Lindsay A Baker / Tessa Sinnige / Pascale Schellenberger / Jeanine de Keyzer / C Alistair Siebert / Arnold J M Driessen / Marc Baldus / Kay Grünewald
Abstract: Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane ...Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted systems.
DateDeposition: Oct 13, 2017 / Header (metadata) release: Dec 20, 2017 / Map release: Dec 27, 2017 / Last update: Jan 10, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3924.map.gz (map file in CCP4 format, 321 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
40 pix
9.04 Å/pix.
= 361.6 Å
50 pix
9.04 Å/pix.
= 452. Å
40 pix
9.04 Å/pix.
= 361.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 9.04 Å
Density
Contour Level:0.01 (by author), 0.01 (movie #1):
Minimum - Maximum-0.04391186 - 0.0651003
Average (Standard dev.)-0.007913827 (0.013773199)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions504040
Origin504040
Limit997979
Spacing405040
CellA: 361.6 Å / B: 452 Å / C: 361.6 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z9.049.049.04
M x/y/z405040
origin x/y/z0.0000.0000.000
length x/y/z361.600452.000361.600
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS405040
NC/NR/NS405040
D min/max/mean-0.0440.065-0.008

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Supplemental data

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Sample components

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Entire Ribosome-nascent chains with native E.coli membranes containing YidC

EntireName: Ribosome-nascent chains with native E.coli membranes containing YidC
Number of components: 3

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Component #1: cellular-component, Ribosome-nascent chains with native E.coli me...

Cellular-componentName: Ribosome-nascent chains with native E.coli membranes containing YidC
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pHisLIC_YidC/pJK763 / Strain: LEMO/BL21delta Trigger Factor
Source (natural)Location in cell: membrane

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Component #2: cellular-component, E. coli native membranes containing YidC

Cellular-componentName: E. coli native membranes containing YidC / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pHisLIC_YidC / Strain: LEMO
Source (natural)Location in cell: membrane

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Component #3: protein, Ribosomes with SecM stalled nascent chains of ATP syntha...

ProteinName: Ribosomes with SecM stalled nascent chains of ATP synthase subunit c
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pJK763 / Strain: BL21 delta Trigger Factor
Source (natural)Location in cell: cytoplasm

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.4
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE-PROPANE MIXTURE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 2500 - 5000 nm / Energy filter: GIF Quantum LS / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt Angle: -45 - 45 deg.
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C1 (asymmetric)
Details: Tomograms were built with IMOD, after correction of movie stacks with Unblur.
3D reconstructionAlgorithm: BACK PROJECTION / Resolution: 100 Å / Resolution method: OTHER

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