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- EMDB-17631: Escherichia coli paused disome complex (queueing 70S non-rotated ... -
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Open data
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Basic information
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Title | Escherichia coli paused disome complex (queueing 70S non-rotated closed PRE state) | |||||||||
![]() | Cryo-EM reconstruction of the E. coli disome complex (local refinement of queueing 70S non-rotated closed PRE state) | |||||||||
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![]() | translation / disome / collision / PURE system / RIBOSOME | |||||||||
Function / homology | ![]() RNA secondary structure unwinding / positive regulation of cytoplasmic translation / negative regulation of cytoplasmic translational initiation / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation ...RNA secondary structure unwinding / positive regulation of cytoplasmic translation / negative regulation of cytoplasmic translational initiation / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / four-way junction DNA binding / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / ribosome assembly / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / transcription elongation factor complex / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / DNA endonuclease activity / ribosomal large subunit assembly / transcription antitermination / response to reactive oxygen species / translational initiation / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / molecular adaptor activity / single-stranded RNA binding / rRNA binding / negative regulation of translation / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
![]() | Fluegel T / Schacherl M | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM. Authors: Timo Flügel / Magdalena Schacherl / Anett Unbehaun / Birgit Schroeer / Marylena Dabrowski / Jörg Bürger / Thorsten Mielke / Thiemo Sprink / Christoph A Diebolder / Yollete V Guillén ...Authors: Timo Flügel / Magdalena Schacherl / Anett Unbehaun / Birgit Schroeer / Marylena Dabrowski / Jörg Bürger / Thorsten Mielke / Thiemo Sprink / Christoph A Diebolder / Yollete V Guillén Schlippe / Christian M T Spahn / ![]() Abstract: Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex ...Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts. | |||||||||
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 1.1 GB | ![]() | |
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Header (meta data) | ![]() ![]() | 80.8 KB 80.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 23.2 KB | Display | ![]() |
Images | ![]() | 146 KB | ||
Masks | ![]() | 1.3 GB | ![]() | |
Filedesc metadata | ![]() | 16.5 KB | ||
Others | ![]() ![]() | 1.2 GB 1.2 GB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8pegMC ![]() 8pklC ![]() 8r3vC ![]() 8rclC ![]() 8rcmC ![]() 8rcsC ![]() 8rctC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Cryo-EM reconstruction of the E. coli disome complex (local refinement of queueing 70S non-rotated closed PRE state) | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
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Projections & Slices |
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Density Histograms |
-Half map: Cryo-EM reconstruction of the E. coli disome complex...
File | emd_17631_half_map_1.map | ||||||||||||
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Annotation | Cryo-EM reconstruction of the E. coli disome complex (local refinement of queueing 70S non-rotated closed PRE state. Half map B) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Cryo-EM reconstruction of the E. coli disome complex...
File | emd_17631_half_map_2.map | ||||||||||||
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Annotation | Cryo-EM reconstruction of the E. coli disome complex (local refinement of queueing 70S non-rotated closed PRE state. Half map A) | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : Escherichia coli paused disome complex
+Supramolecule #1: Escherichia coli paused disome complex
+Macromolecule #1: 50S ribosomal protein L36
+Macromolecule #2: 50S ribosomal protein L28
+Macromolecule #3: 50S ribosomal protein L29
+Macromolecule #4: 50S ribosomal protein L30
+Macromolecule #5: Large ribosomal subunit protein bL31A
+Macromolecule #6: 50S ribosomal protein L32
+Macromolecule #7: 50S ribosomal protein L35
+Macromolecule #11: Small ribosomal subunit protein uS2
+Macromolecule #12: Small ribosomal subunit protein uS3
+Macromolecule #13: Small ribosomal subunit protein uS4
+Macromolecule #14: Small ribosomal subunit protein uS5
+Macromolecule #15: 30S ribosomal protein S6
+Macromolecule #16: 30S ribosomal protein S7
+Macromolecule #17: Small ribosomal subunit protein uS8
+Macromolecule #18: Small ribosomal subunit protein uS9
+Macromolecule #19: 30S ribosomal protein S10
+Macromolecule #20: Small ribosomal subunit protein uS11
+Macromolecule #21: Small ribosomal subunit protein uS12
+Macromolecule #22: Small ribosomal subunit protein uS13
+Macromolecule #23: Small ribosomal subunit protein uS14
+Macromolecule #24: 30S ribosomal protein S15
+Macromolecule #25: 30S ribosomal protein S16
+Macromolecule #26: Small ribosomal subunit protein uS17
+Macromolecule #27: Small ribosomal subunit protein bS18
+Macromolecule #28: Small ribosomal subunit protein uS19
+Macromolecule #29: Small ribosomal subunit protein bS20
+Macromolecule #30: 30S ribosomal protein S21
+Macromolecule #35: 30S ribosomal protein S1
+Macromolecule #36: Large ribosomal subunit protein uL1
+Macromolecule #37: 50S ribosomal protein L2
+Macromolecule #38: 50S ribosomal protein L3
+Macromolecule #39: 50S ribosomal protein L4
+Macromolecule #40: 50S ribosomal protein L5
+Macromolecule #41: 50S ribosomal protein L6
+Macromolecule #42: 50S ribosomal protein L33
+Macromolecule #43: 50S ribosomal protein L16
+Macromolecule #44: 50S ribosomal protein L9
+Macromolecule #45: Large ribosomal subunit protein uL10
+Macromolecule #46: 50S ribosomal protein L11
+Macromolecule #47: 50S ribosomal protein L34
+Macromolecule #48: 50S ribosomal protein L13
+Macromolecule #49: 50S ribosomal protein L14
+Macromolecule #50: 50S ribosomal protein L15
+Macromolecule #51: Nascent chain
+Macromolecule #52: 50S ribosomal protein L17
+Macromolecule #53: 50S ribosomal protein L18
+Macromolecule #54: 50S ribosomal protein L19
+Macromolecule #55: 50S ribosomal protein L20
+Macromolecule #56: 50S ribosomal protein L21
+Macromolecule #57: 50S ribosomal protein L22
+Macromolecule #58: 50S ribosomal protein L23
+Macromolecule #59: 50S ribosomal protein L24
+Macromolecule #60: 50S ribosomal protein L25
+Macromolecule #61: 50S ribosomal protein L27
+Macromolecule #8: 23S ribosomal RNA
+Macromolecule #9: 5S ribosomal RNA
+Macromolecule #10: 16S ribosomal RNA
+Macromolecule #31: mRNA
+Macromolecule #32: tRNA-Phe (P-site)
+Macromolecule #33: tRNA-Ser (E-site)
+Macromolecule #34: tRNA-Val (A-site)
+Macromolecule #62: ZINC ION
+Macromolecule #63: MAGNESIUM ION
+Macromolecule #64: ADENOSINE-5'-TRIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.6 |
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Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.037 kPa Details: Operated at 15 mA, easiGlow Discharge Cleaning system (PELCO) |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: Withdrawn samples were spotted directly onto freshly glow-discharged holey carbon grids, blotted for 1-2 s, and flash frozen in liquid ethane using a Vitrobot Mark IV plunger (ThermoFisher ...Details: Withdrawn samples were spotted directly onto freshly glow-discharged holey carbon grids, blotted for 1-2 s, and flash frozen in liquid ethane using a Vitrobot Mark IV plunger (ThermoFisher Scientific) after a wait time of 40 s at 4 degrees Celcius.. |
Details | In vitro translation reactions were performed in the PURE translation system using the PURExpress delta ribosome kit (NEB, #E3313S). Translation reactions were supplemented with 0.8 U/uL RNAsin Plus RNase Inhibitor (Promega, N261B). SolA, factor mix, and RNAsin Plus were combined on ice, followed by a preincubation at 37 degrees Celcius for 2 min, and added directly to polysomes (700 nM final concentration) that had been preincubated at 37 degrees Celsius for 2 min. After 1 min reaction time, 4 uL of the reaction mixture were withdrawn for plunge freezing. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 80.0 K / Max: 82.0 K |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8982 / Average exposure time: 1.13 sec. / Average electron dose: 45.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Details | The atomic model of the E. coli 70S ribosome (PDB ID: 7N1P) was initially docked into the postprocessed density maps with UCSF Chimera and manually adjusted in Coot. All models were refined over multiple rounds using the module phenix.real_space_refine in PHENIX and interactive model building and refinement in Coot, using libG restraints for the RNAs. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: CC |
Output model | ![]() PDB-8peg: |