|Entry||Database: EMDB / ID: 2976|
|Title||Time-resolved Cryo Electron Microscopy of ribosome subunit association|
|Map data||Reconstruction of E. Coli naked 70S ribosome in non-rotated (NR) conformation|
|Sample||E. Coli 70S Ribosome:|
|Keywords||time-resolved / cryo-EM / mixing-spraying / ribosome subunit association / structural dynamics|
|Source||Escherichia coli (E. coli)|
|Method||single particle reconstruction / cryo EM / 9.7 Å resolution|
|Authors||Chen B / Kaledhonkar S / Sun M / Shen B / Lu Z / Barnard D / Lu T / Gonzalez Jr R / Frank J|
|Citation||Journal: 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.
|Date||Deposition: Apr 7, 2015 / Header (metadata) release: Jun 17, 2015 / Map release: Jun 17, 2015 / Last update: Jun 17, 2015|
|Structure viewer||EM map: |
Downloads & links
|File||emd_2976.map.gz (map file in CCP4 format, 16001 KB)|
|Projections & slices|
Images are generated by Spider.
CCP4 map header:
-Entire E. Coli 70S Ribosome
|Entire||Name: E. Coli 70S Ribosome / Number of components: 1|
-Component #1: ribosome-prokaryote, Escherichia coli 70S ribosome
|Ribosome-prokaryote||Name: Escherichia coli 70S ribosome / Prokaryote: LSU 50S, SSU 30S / Recombinant expression: No|
|Source||Species: Escherichia coli (E. coli) / Strain: MRE600|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Buffer solution: 25 mM Tris-HCl, 60 mM NH4Cl, 5 mM 2-mercaptoethanol, 3.5 mM MgCl2|
|Support film||Quantifoil R2/2 300 mesh copper grid with thin carbon sipport|
|Vitrification||Instrument: 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).
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20 / Date: Sep 13, 2013 / Details: Low dose, Data was collected over two years time|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 17 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 50000 X (nominal), 66318 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 2000 - 4000 nm|
|Specimen Holder||Model: GATAN LIQUID NITROGEN / Temperature: 80 K|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Image acquisition||Number of digital images: 3402|
|Processing||Method: 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 reconstruction||Software: Arachnid, RELION, SPIDER / CTF correction: each Micrograph / Resolution: 9.7 Å / Resolution method: FSC 0.143, gold-standard|
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