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- EMDB-8115: Structure of RelA bound to the 70S ribosome (focused classificati... -

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Basic information

Entry
Database: EMDB / ID: EMD-8115
TitleStructure of RelA bound to the 70S ribosome (focused classification, RRM)
Map dataNone
Sample
  • Complex: 70 S ribosome
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 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
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  • 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

emd_8115.png

Downloads & links

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Map

FileDownload / File: emd_8115.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.55483943 - 1.1531812
Average (Standard dev.)0.0014773944 (±0.024344739)
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-0.5551.1530.001

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Supplemental data

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Mask #1

Fileemd_8115_msk_1.map
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Mask #2

Fileemd_8115_msk_2.map
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Additional map: None

Fileemd_8115_additional.map
AnnotationNone
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Half map: None

Fileemd_8115_half_map_1.map
AnnotationNone
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Half map: None

Fileemd_8115_half_map_2.map
AnnotationNone
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Sample components

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Entire : 70 S ribosome

EntireName: 70 S ribosome
Components
  • Complex: 70 S ribosome

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Supramolecule #1: 70 S ribosome

SupramoleculeName: 70 S ribosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#59
Source (natural)Organism: Escherichia coli (E. coli)

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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

Concentration0.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
5.0 mMHepes
50.0 mMKCl
10.0 mMNH4Cl
10.0 mMMg(OAc)2
6.0 mMbeta-mercaptoethanol
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II / Details: Grids were blotted for 5 s.
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: OTHER / Average electron dose: 35.0 e/Å2
Details: FEI Falcon III Images were collected in movie-mode at 30 frames per second
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 183615
CTF correctionSoftware - Name: Gctf (ver. 0.5)
Startup modelType of model: EMDB MAP / Details: EMDB-2373
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4)
Details: Refinement of particles selected using focused classification with signal subtraction (FCwSS) with a mask over the RelA RNA Recognition Motif (RRM).
Number images used: 86985
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4ybb

RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 41.3 / Target criteria: Average FSC

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