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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: 2976
TitleTime-resolved Cryo Electron Microscopy of ribosome subunit association
Map dataReconstruction of E. Coli naked 70S ribosome in non-rotated (NR) conformation
SampleE. Coli 70S Ribosome:
ribosome-prokaryote
Keywordstime-resolved / cryo-EM / mixing-spraying / ribosome subunit association / structural dynamics
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 9.7 Å resolution
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.
DateDeposition: Apr 7, 2015 / Header (metadata) release: Jun 17, 2015 / Map release: Jun 17, 2015 / Last update: Jun 17, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • 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

Downloads & links

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Map

Fileemd_2976.map.gz (map file in CCP4 format, 16001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
160 pix
1 Å/pix.
= 160. Å
160 pix
1 Å/pix.
= 160. Å
160 pix
1 Å/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
Contour Level:0.04 (by author), 0.04 (movie #1):
Minimum - Maximum-0.09787601 - 0.19526787
Average (Standard dev.)0.00143119 (0.02871864)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions160160160
Origin000
Limit159159159
Spacing160160160
CellA=B=C: 160 Å
α=β=γ: 90 deg.

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 / Number of components: 1

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Component #1: ribosome-prokaryote, Escherichia coli 70S ribosome

Ribosome-prokaryoteName: Escherichia coli 70S ribosome / Prokaryote: LSU 50S, SSU 30S / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: MRE600

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionBuffer solution: 25 mM Tris-HCl, 60 mM NH4Cl, 5 mM 2-mercaptoethanol, 3.5 mM MgCl2
pH: 7.6
Support filmQuantifoil R2/2 300 mesh copper grid with thin carbon sipport
VitrificationInstrument: OTHER / Cryogen name: ETHANE / Temperature: 80 K / Humidity: 80 % / Time resolved state: Vitrified after spraying
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).

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Sep 13, 2013 / Details: Low dose, Data was collected over two years time
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 17 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal), 66318 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 2000 - 4000 nm
Specimen HolderModel: GATAN LIQUID NITROGEN / Temperature: 80 K
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3402

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 39678
Details: The partciles were selected with Autopicker (Langlois et al., 2014), and 3D classification and reconstruction with RELION
3D reconstructionSoftware: Arachnid, RELION, SPIDER / CTF correction: each Micrograph / Resolution: 9.7 Å / Resolution method: FSC 0.143, gold-standard

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