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- EMDB-3639: 100S disome from Staphylococcus aureus (Loose conformation) -

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Basic information

Entry
Database: EMDB / ID: EMD-3639
Title100S disome from Staphylococcus aureus (Loose conformation)
Map data
Sample
  • Complex: 100S disome from Staphylococcus aureus (Loose conformation)
    • Complex: S. aureus 100S ribosome
    • Complex: Ribosome hibernation promoting factor (SaHPF)
Biological speciesStaphylococcus aureus (strain NCTC 8325) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsKhusainov I / Vicens Q / Ayupov R / Usachev K / Myasnikov A / Simonetti A / Validov S / Kieffer B / Yusupova G / Yusupov M / Hashem Y
CitationJournal: EMBO J / Year: 2017
Title: Structures and dynamics of hibernating ribosomes from mediated by intermolecular interactions of HPF.
Authors: Iskander Khusainov / Quentin Vicens / Rustam Ayupov / Konstantin Usachev / Alexander Myasnikov / Angelita Simonetti / Shamil Validov / Bruno Kieffer / Gulnara Yusupova / Marat Yusupov / Yaser Hashem /
Abstract: In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of ...In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen containing a long variant of the hibernation-promoting factor (SaHPF) that we solved using cryo-electron microscopy. Our reconstructions reveal that the N-terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C-terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD-dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.
History
DepositionMar 17, 2017-
Header (metadata) releaseMar 29, 2017-
Map releaseJun 28, 2017-
UpdateAug 2, 2017-
Current statusAug 2, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.076
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.076
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3639.png

Downloads & links

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Map

FileDownload / File: emd_3639.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.3 Å/pix.
x 200 pix.
= 660. Å		3.3 Å/pix.
x 200 pix.
= 660. Å		3.3 Å/pix.
x 200 pix.
= 660. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.076 / Movie #1: 0.076
Minimum - Maximum-0.16953567 - 0.36963096
Average (Standard dev.)0.0011841003 (±0.02209469)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 660.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.33.33.3
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z660.000660.000660.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1700.3700.001

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Supplemental data

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Sample components

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Entire : 100S disome from Staphylococcus aureus (Loose conformation)

EntireName: 100S disome from Staphylococcus aureus (Loose conformation)
Components
  • Complex: 100S disome from Staphylococcus aureus (Loose conformation)
    • Complex: S. aureus 100S ribosome
    • Complex: Ribosome hibernation promoting factor (SaHPF)

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Supramolecule #1: 100S disome from Staphylococcus aureus (Loose conformation)

SupramoleculeName: 100S disome from Staphylococcus aureus (Loose conformation)
type: complex / ID: 1 / Parent: 0

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Supramolecule #2: S. aureus 100S ribosome

SupramoleculeName: S. aureus 100S ribosome / type: complex / ID: 2 / Parent: 1 / Details: Two vacant 70S ribosomes dimerized via SaHPF
Source (natural)Organism: Staphylococcus aureus (strain NCTC 8325) (bacteria)

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Supramolecule #3: Ribosome hibernation promoting factor (SaHPF)

SupramoleculeName: Ribosome hibernation promoting factor (SaHPF) / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Staphylococcus aureus (strain NCTC 8325) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)

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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

BufferpH: 7.5
Details: 5 mM Hepes-KOH pH 7.5 50 mM KCl 10 mM NH4Cl 10 mM Mg(OAc)2 1 mM DTT
GridMaterial: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 5, blot waiting time 30 s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-8 / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 132000
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

4050

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 17300
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final 3D classificationSoftware - Name: RELION (ver. 1.3)

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