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- EMDB-2976: Time-resolved Cryo Electron Microscopy of ribosome subunit association -

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Basic information

Entry
Database: EMDB / ID: EMD-2976
TitleTime-resolved Cryo Electron Microscopy of ribosome subunit association
Map dataReconstruction of E. Coli naked 70S ribosome in non-rotated (NR) conformation
Sample
  • Sample: E. Coli 70S Ribosome
  • Complex: Escherichia coli 70S ribosome
Keywordstime-resolved / cryo-EM / mixing-spraying / ribosome subunit association / structural dynamics
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.7 Å
AuthorsChen B / Kaledhonkar S / Sun M / Shen B / Lu Z / Barnard D / Lu T / Gonzalez Jr R / Frank J
CitationJournal: Structure / Year: 2015
Title: Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy.
Authors: Bo Chen / Sandip Kaledhonkar / Ming Sun / Bingxin Shen / Zonghuan Lu / David Barnard / Toh-Ming Lu / Ruben L Gonzalez / Joachim Frank /
Abstract: Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, ...Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, and ribosome recycling, are amenable to study with this method.
History
DepositionApr 7, 2015-
Header (metadata) releaseJun 17, 2015-
Map releaseJun 17, 2015-
UpdateJun 17, 2015-
Current statusJun 17, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2976.png

Downloads & links

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Map

FileDownload / File: emd_2976.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of E. Coli naked 70S ribosome in non-rotated (NR) conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 160 pix.
= 160. Å		1 Å/pix.
x 160 pix.
= 160. Å		1 Å/pix.
x 160 pix.
= 160. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.111
CCP4 map header111
EM Navigator Movie #12.252.252.25
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.09787601 - 0.19526787
Average (Standard dev.)0.00143119 (±0.02871864)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 160.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z160.000160.000160.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0980.1950.001

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Supplemental data

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Sample components

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Entire : E. Coli 70S Ribosome

EntireName: E. Coli 70S Ribosome
Components
  • Sample: E. Coli 70S Ribosome
  • Complex: Escherichia coli 70S ribosome

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Supramolecule #1000: E. Coli 70S Ribosome

SupramoleculeName: E. Coli 70S Ribosome / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Escherichia coli 70S ribosome

SupramoleculeName: Escherichia coli 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S, SSU 30S
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600

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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: 7.6
Details: 25 mM Tris-HCl, 60 mM NH4Cl, 5 mM 2-mercaptoethanol, 3.5 mM MgCl2
GridDetails: Quantifoil R2/2 300 mesh copper grid with thin carbon sipport
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 80 K / Instrument: OTHER
Details: Equal volume of 1.2 microM 30S and 0.6 microM 50S (final concentration after mixing) were injected into the mixing-spraying device each at flow rate of 3 microL/s. The computer-controlled ...Details: Equal volume of 1.2 microM 30S and 0.6 microM 50S (final concentration after mixing) were injected into the mixing-spraying device each at flow rate of 3 microL/s. The computer-controlled plunging device was purchased from Dr. Howard White (Eastern Virginia Medical School, VA).
Timed resolved state: Vitrified after spraying

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 80 K
DetailsLow dose, Data was collected over two years time
DateSep 13, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 3402 / Average electron dose: 17 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 66318 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe partciles were selected with Autopicker (Langlois et al., 2014), and 3D classification and reconstruction with RELION
CTF correctionDetails: each Micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.7 Å / Resolution method: OTHER / Software - Name: Arachnid, RELION, SPIDER / Number images used: 39678

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