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- EMDB-8411: Time-resolved cryo electron microscopy map of the RRF-bound post-... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-8411 | |||||||||||||||
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Title | Time-resolved cryo electron microscopy map of the RRF-bound post-termination ribosome complex | |||||||||||||||
![]() | 70S ribosome, RRF, P/E site tRNA, mRNA, (Rot) | |||||||||||||||
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Biological species | ![]() ![]() | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 10.0 Å | |||||||||||||||
![]() | Fu Z / Kaledhonkar S / Borg A / Sun M / Chen B / Grassucci RA / Ehrenberg M / Frank J | |||||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 198.7 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.2 KB 14.2 KB | Display Display | ![]() |
Images | ![]() | 66.3 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8412C ![]() 8413C ![]() 8415C ![]() 8416C ![]() 8417C ![]() 8418C C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 70S ribosome, RRF, P/E site tRNA, mRNA, (Rot) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
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Sample components
-Entire : 70S ribosome-RRF-tRNA-mRNA complex
Entire | Name: 70S ribosome-RRF-tRNA-mRNA complex |
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Components |
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-Supramolecule #1: 70S ribosome-RRF-tRNA-mRNA complex
Supramolecule | Name: 70S ribosome-RRF-tRNA-mRNA complex / type: complex / ID: 1 / Parent: 0 |
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Molecular weight | Theoretical: 2.7 MDa |
-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: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Supramolecule #3: P/E site tRNA Phenylalanine
Supramolecule | Name: P/E site tRNA Phenylalanine / type: complex / ID: 3 / Parent: 1 |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Supramolecule #4: mRNA
Supramolecule | Name: mRNA / type: complex / ID: 4 / Parent: 1 Details: Encodes the peptide fMet-Phe-Thr (sequence GGGAAUUCGGGCCCUUGUUAACAAUUAAGGAGGUAUUAAAUGUUUACGUAAUUGCAGAAAAAAAAAAAAAAAAAAAAA) |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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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 |
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Electron microscopy
Microscope | FEI TECNAI F30 |
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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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 31000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Tecnai F30 / Image courtesy: FEI Company |
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Image processing
CTF correction | Software - Name: CTFFIND3 |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 11187 |
Initial angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3) |
Final angle assignment | Type: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3) |