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Yorodumi- EMDB-8416: Time-resolved cryo electron microscopy map of the EF-G-, RRF-, an... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8416 | |||||||||||||||
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Title | Time-resolved cryo electron microscopy map of the EF-G-, RRF-, and tRNA-bound 50S subunit | |||||||||||||||
Map data | RRF, EF-G and tRNA bound 50S subunit | |||||||||||||||
Sample |
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Biological species | Escherichia coli (E. coli) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 16.0 Å | |||||||||||||||
Authors | Fu Z / Kaledhonkar S / Borg A / Sun M / Chen B / Grassucci RA / Ehrenberg M / Frank J | |||||||||||||||
Funding support | United States, Sweden, 4 items
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Citation | Journal: Structure / Year: 2016 Title: Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy. Authors: Ziao Fu / Sandip Kaledhonkar / Anneli Borg / Ming Sun / Bo Chen / Robert A Grassucci / Måns Ehrenberg / Joachim Frank / Abstract: Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination ...Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures. | |||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8416.map.gz | 196.2 MB | EMDB map data format | |
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Header (meta data) | emd-8416-v30.xml emd-8416.xml | 14.3 KB 14.3 KB | Display Display | EMDB header |
Images | emd_8416.png | 46.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8416 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8416 | HTTPS FTP |
-Related structure data
Related structure data | 8411C 8412C 8413C 8415C 8417C 8418C C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_8416.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | RRF, EF-G and tRNA bound 50S subunit | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.25 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : RRF, EF-G-, and tRNA-bound 50S subunit
Entire | Name: RRF, EF-G-, and tRNA-bound 50S subunit |
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Components |
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-Supramolecule #1: RRF, EF-G-, and tRNA-bound 50S subunit
Supramolecule | Name: RRF, EF-G-, and tRNA-bound 50S subunit / type: complex / ID: 1 / Parent: 0 |
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-Supramolecule #2: RRF
Supramolecule | Name: RRF / type: complex / ID: 2 / Parent: 1 Details: His-tagged RRF purified by nickel affinity chromatography. |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Supramolecule #3: tRNA
Supramolecule | Name: tRNA / type: complex / ID: 3 / Parent: 1 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Supramolecule #4: EF-G
Supramolecule | Name: EF-G / type: complex / ID: 4 / Parent: 1 / Details: EF-G purified by nickel affinity chromatography |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2.7 mg/mL |
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Buffer | pH: 7.5 |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER |
-Electron microscopy
Microscope | FEI TECNAI F30 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 31000 |
Specialist optics | Spherical aberration corrector: None / Chromatic aberration corrector: None / Energy filter - Name: None |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN |
Temperature | Min: 80.0 K |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Average exposure time: 0.2 sec. / Average electron dose: 1.01 e/Å2 |
Experimental equipment | Model: Tecnai F30 / Image courtesy: FEI Company |
-Image processing
CTF correction | Software - Name: CTFFIND3 |
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Initial angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3) |
Final angle assignment | Type: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3) |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 8059 |