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- EMDB-3919: Stalled E. coli ribosomes (Fo-c SecM nascent chains) and native E... -

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Basic information

Entry
Database: EMDB / ID: EMD-3919
TitleStalled E. coli ribosomes (Fo-c SecM nascent chains) and native E. coli membranes containing recombinant YidC
Map dataAverage of class 1
Sample
  • Organelle or cellular component: Ribosome-nascent chains with native E.coli membranes containing YidC
    • Organelle or cellular component: E. coli native membranes containing YidC
    • Complex: Ribosomes with SecM stalled nascent chains of ATP synthase subunit c
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 100.0 Å
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.
History
DepositionOct 12, 2017-
Header (metadata) releaseDec 20, 2017-
Map releaseDec 27, 2017-
UpdateJan 10, 2018-
Current statusJan 10, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0055
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0055
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3919.png

Downloads & links

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Map

FileDownload / File: emd_3919.map.gz / Format: CCP4 / Size: 313.5 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAverage of class 1
Projections & slices

Image control

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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 9.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0055 / Movie #1: 0.0055
Minimum - Maximum-0.03396017 - 0.034291007
Average (Standard dev.)-0.00652426 (±0.01028893)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin504040
Dimensions504040
Spacing405040
CellA: 361.6 Å / B: 452.0 Å / C: 361.6 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS405040
NC/NR/NS405040
D min/max/mean-0.0340.034-0.007

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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
Components
  • Organelle or cellular component: Ribosome-nascent chains with native E.coli membranes containing YidC
    • Organelle or cellular component: E. coli native membranes containing YidC
    • Complex: Ribosomes with SecM stalled nascent chains of ATP synthase subunit c

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Supramolecule #1: Ribosome-nascent chains with native E.coli membranes containing YidC

SupramoleculeName: Ribosome-nascent chains with native E.coli membranes containing YidC
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: membrane
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: LEMO/BL21delta Trigger Factor / Recombinant plasmid: pHisLIC_YidC/pJK763

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Supramolecule #2: E. coli native membranes containing YidC

SupramoleculeName: E. coli native membranes containing YidC / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: membrane
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: LEMO / Recombinant plasmid: pHisLIC_YidC

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Supramolecule #3: Ribosomes with SecM stalled nascent chains of ATP synthase subunit c

SupramoleculeName: Ribosomes with SecM stalled nascent chains of ATP synthase subunit c
type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 delta Trigger Factor / Recombinant plasmid: pJK763

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3836 pixel / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 8.0 µm / Calibrated defocus min: 4.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsTomograms were built with IMOD, after correction of movie stacks with Unblur.
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 100.0 Å / Resolution method: OTHER / Number subtomograms used: 75
ExtractionNumber tomograms: 47 / Number images used: 396
CTF correctionSoftware - Name: IMOD (ver. 4.9.0)
Final 3D classificationNumber classes: 10 / Software - Name: PEET (ver. 1.10.0)
Final angle assignmentType: NOT APPLICABLE

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