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- EMDB-8108: Structure of RelA bound to the 70S ribosome (overall map, class 2) -

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Basic information

Entry
Database: EMDB / ID: EMD-8108
TitleStructure of RelA bound to the 70S ribosome (overall map, class 2)
Map data
Sample70 S ribosome
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsBrown A / Fernandez IS / Gordiyenko Y / Ramakrishnan V
CitationJournal: Nature / Year: 2016
Title: Ribosome-dependent activation of stringent control.
Authors: Alan Brown / Israel S Fernández / Yuliya Gordiyenko / V Ramakrishnan /
Abstract: In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid ...In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.
History
DepositionMar 11, 2016-
Header (metadata) releaseJun 29, 2016-
Map releaseJun 29, 2016-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8108.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 400 pix.
= 536. Å
1.34 Å/pix.
x 400 pix.
= 536. Å
1.34 Å/pix.
x 400 pix.
= 536. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.6347005 - 1.1297468
Average (Standard dev.)0.0014723118 (±0.025488678)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 536.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z536.000536.000536.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-41
NX/NY/NZ737384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.6351.1300.001

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Supplemental data

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Half map: None

Fileemd_8108_half_map_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: None

Fileemd_8108_half_map_2.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire 70 S ribosome

EntireName: 70 S ribosome / Number of components: 1

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Component #1: protein, 70 S ribosome

ProteinName: 70 S ribosome / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.1 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 % / Details: Grids were blotted for 5 s.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 104478.0 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: 1800.0 - 3000.0 nm
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image acquisition

Image acquisitionDetails: FEI Falcon III Images were collected in movie-mode at 30 frames per second

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 98498
3D reconstructionSoftware: RELION / Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: Average FSC / Refinement space: RECIPROCAL
Input PDB model: 4YBB
Overall bvalue: 112.7

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