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- PDB-8rcs: Escherichia coli paused disome complex (Rotated disome interface ... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8rcs | ||||||
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Title | Escherichia coli paused disome complex (Rotated disome interface class 1) | ||||||
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Function / homology | ![]() RNA secondary structure unwinding / positive regulation of cytoplasmic translation / negative regulation of cytoplasmic translational initiation / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / positive regulation of ribosome biogenesis ...RNA secondary structure unwinding / positive regulation of cytoplasmic translation / negative regulation of cytoplasmic translational initiation / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / negative regulation of translational initiation / translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / transcription elongation factor complex / cytosolic ribosome assembly / DNA endonuclease activity / regulation of DNA-templated transcription elongation / transcription antitermination / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() | ||||||
Method | ![]() ![]() ![]() | ||||||
![]() | 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. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 4.1 MB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 19058MC ![]() 8pegC ![]() 8pklC ![]() 8r3vC ![]() 8rclC ![]() 8rcmC ![]() 8rctC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Components
-50S ribosomal protein ... , 12 types, 12 molecules 123262b2e2g2h2i2l2o2r2z2
#1: Protein | ![]() Mass: 9027.551 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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#2: Protein | ![]() Mass: 6554.820 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#4: Protein | ![]() Mass: 7313.032 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#35: Protein | ![]() Mass: 29923.619 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#36: Protein | ![]() Mass: 20333.611 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#37: Protein | ![]() Mass: 6388.631 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#38: Protein | ![]() Mass: 15312.269 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#39: Protein | ![]() Mass: 15789.020 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#40: Protein/peptide | ![]() Mass: 5397.463 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#41: Protein | ![]() Mass: 15008.471 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#43: Protein | ![]() Mass: 12794.668 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#44: Protein | ![]() Mass: 9146.540 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
-Large ribosomal subunit protein ... , 2 types, 3 molecules 441a2
#3: Protein | Mass: 7887.117 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #34: Protein | | Mass: 24765.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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-RNA chain , 8 types, 10 molecules 717282A1A2V2WW1YY1
#5: RNA chain | ![]() Mass: 941812.562 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #6: RNA chain | | ![]() Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() ![]() #7: RNA chain | ![]() Mass: 499873.406 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() ![]() #28: RNA chain | | ![]() Mass: 19140.500 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #29: RNA chain | | Mass: 24628.865 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #30: RNA chain | | Mass: 24651.904 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #31: RNA chain | | Mass: 24555.654 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #32: RNA chain | | Mass: 24617.768 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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-Small ribosomal subunit protein ... , 14 types, 27 molecules BB2C1C2D1D2E1E2H1H2I1I2K1K2L1L2M1M2N1N2Q1Q2R1R2S1S2T1
#8: Protein | Mass: 26781.670 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #9: Protein | Mass: 26031.316 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #10: Protein | Mass: 23514.199 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #11: Protein | Mass: 17629.398 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #14: Protein | Mass: 14146.557 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #15: Protein | Mass: 14886.270 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #17: Protein | Mass: 13870.975 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #18: Protein | Mass: 13768.157 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #19: Protein | Mass: 13128.467 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #20: Protein | Mass: 11606.560 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #23: Protein | Mass: 9724.491 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #24: Protein | Mass: 9005.472 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #25: Protein | Mass: 10455.355 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #26: Protein | | Mass: 9708.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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-30S ribosomal protein ... , 7 types, 12 molecules F1F2G1G2J1J2O1O2P1U1U2Z1
#12: Protein | ![]() Mass: 15727.512 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #13: Protein | ![]() Mass: 20055.156 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #16: Protein | ![]() Mass: 11755.597 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #21: Protein | ![]() Mass: 10290.816 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #22: Protein | | ![]() Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #27: Protein | ![]() Mass: 8524.039 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #33: Protein | | ![]() Mass: 61238.887 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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-Protein/peptide , 1 types, 1 molecules p
#42: Protein/peptide | Mass: 1087.235 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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-Non-polymers , 4 types, 274 molecules ![](data/chem/img/ZN.gif)
![](data/chem/img/PUT.gif)
![](data/chem/img/SPD.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/PUT.gif)
![](data/chem/img/SPD.gif)
![](data/chem/img/MG.gif)
#45: Chemical | #46: Chemical | ![]() #47: Chemical | ChemComp-SPD / | ![]() #48: Chemical | ChemComp-MG / |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: Escherichia coli paused disome complex / Type: RIBOSOME / Details: Structure of the rotated disome interface class 1 / Entity ID: #1-#44 / Source: NATURAL |
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Molecular weight | Value: 2.270379 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() ![]() |
Buffer solution | pH: 7.6 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() 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 ...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. |
Specimen support | Details: Operated at 15 mA, easiGlow Discharge Cleaning system (PELCO) Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification![]() | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 75 % / Chamber temperature: 277 K 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. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() ![]() |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 82 K / Temperature (min): 80 K |
Image recording | Average exposure time: 1.13 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8982 |
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Processing
EM software |
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CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1883839 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 4.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10748 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient 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 ...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. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 7N1P Accession code: 7N1P / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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