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- EMDB-19096: Escherichia coli paused disome complex (local refinement of leadi... -
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Open data
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Basic information
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Title | Escherichia coli paused disome complex (local refinement of leading 70S rotated PRE-1) | |||||||||
![]() | Cryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S rotated PRE-1). Full map. | |||||||||
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![]() | ribosome / polysome / tranlsation / elongation / pausing / disome / collision / PURE system | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.45 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 | ![]() | 186.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 25 KB 25 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 15.4 KB | Display | ![]() |
Images | ![]() | 53.1 KB | ||
Masks | ![]() | 391 MB | ![]() | |
Filedesc metadata | ![]() | 5 KB | ||
Others | ![]() ![]() | 362.3 MB 362.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | Cryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S rotated PRE-1). Full map. | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.59 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_19096_half_map_1.map | ||||||||||||
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Annotation | Cryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S rotated PRE-1). Half map A. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Cryo-EM reconstruction of the E. coli disome complex...
File | emd_19096_half_map_2.map | ||||||||||||
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Annotation | Cryo-EM reconstruction of the E. coli disome complex (local refinement of leading 70S rotated PRE-1). Half map B. | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Escherichia coli paused disome complex
Entire | Name: Escherichia coli paused disome complex |
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Components |
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-Supramolecule #1: Escherichia coli paused disome complex
Supramolecule | Name: Escherichia coli paused disome complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#33, #35-#60 Details: Model of leading ribosome, focused refinement on leading ribosome applied on disome map |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 2.270379 MDa |
-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 |
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 |
Software | Name: EPU (ver. 2.8.1) |
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
Software | Name: ![]() |
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