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Open data
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Basic information
Entry | Database: PDB / ID: 6hrm | |||||||||||||||
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Title | E. coli 70S d2d8 stapled ribosome | |||||||||||||||
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![]() | RIBOSOME / staple | |||||||||||||||
Function / homology | ![]() negative regulation of cytoplasmic translational initiation / stringent response / 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 / transcriptional attenuation / endoribonuclease inhibitor activity ...negative regulation of cytoplasmic translational initiation / stringent response / 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 / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / four-way junction DNA binding / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / translational initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / transcription elongation factor complex / DNA endonuclease activity / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / transcription antitermination / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / ribosomal large subunit assembly / 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 / tRNA binding / molecular adaptor activity / cytoplasmic translation / 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 | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å | |||||||||||||||
![]() | Schmied, W.H. / Tnimov, Z. / Uttamapinant, C. / Rae, C.D. / Fried, S.D. / Chin, J.W. | |||||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() Title: Controlling orthogonal ribosome subunit interactions enables evolution of new function. Authors: Wolfgang H Schmied / Zakir Tnimov / Chayasith Uttamapinant / Christopher D Rae / Stephen D Fried / Jason W Chin / ![]() ![]() ![]() Abstract: Orthogonal ribosomes are unnatural ribosomes that are directed towards orthogonal messenger RNAs in Escherichia coli, through an altered version of the 16S ribosomal RNA of the small subunit. ...Orthogonal ribosomes are unnatural ribosomes that are directed towards orthogonal messenger RNAs in Escherichia coli, through an altered version of the 16S ribosomal RNA of the small subunit. Directed evolution of orthogonal ribosomes has provided access to new ribosomal function, and the evolved orthogonal ribosomes have enabled the encoding of multiple non-canonical amino acids into proteins. The original orthogonal ribosomes shared the pool of 23S ribosomal RNAs, contained in the large subunit, with endogenous ribosomes. Selectively directing a new 23S rRNA to an orthogonal mRNA, by controlling the association between the orthogonal 16S rRNAs and 23S rRNAs, would enable the evolution of new function in the large subunit. Previous work covalently linked orthogonal 16S rRNA and a circularly permuted 23S rRNA to create orthogonal ribosomes with low activity; however, the linked subunits in these ribosomes do not associate specifically with each other, and mediate translation by associating with endogenous subunits. Here we discover engineered orthogonal 'stapled' ribosomes (with subunits linked through an optimized RNA staple) with activities comparable to that of the parent orthogonal ribosome; they minimize association with endogenous subunits and mediate translation of orthogonal mRNAs through the association of stapled subunits. We evolve cells with genomically encoded stapled ribosomes as the sole ribosomes, which support cellular growth at similar rates to natural ribosomes. Moreover, we visualize the engineered stapled ribosome structure by cryo-electron microscopy at 3.0 Å, revealing how the staple links the subunits and controls their association. We demonstrate the utility of controlling subunit association by evolving orthogonal stapled ribosomes which efficiently polymerize a sequence of monomers that the natural ribosome is intrinsically unable to translate. Our work provides a foundation for evolving the rRNA of the entire orthogonal ribosome for the encoded cellular synthesis of non-canonical biological polymers. | |||||||||||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 3.2 MB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.3 MB | Display | ![]() |
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Full document | ![]() | 1.4 MB | Display | |
Data in XML | ![]() | 226.5 KB | Display | |
Data in CIF | ![]() | 384 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 0261MC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Components
-RNA chain , 2 types, 2 molecules 13
#1: RNA chain | Mass: 1445685.000 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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#2: RNA chain | Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
+50S ribosomal protein ... , 31 types, 31 molecules BCDEFGHIJKLMNOPQRSTUVWXYZabcdef
-30S ribosomal protein ... , 20 types, 20 molecules ghijklmnopqrstuvwxyz
#34: Protein | Mass: 25072.867 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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#35: Protein | Mass: 23248.994 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#36: Protein | Mass: 23383.002 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#37: Protein | Mass: 16475.037 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#38: Protein | Mass: 12125.993 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#39: Protein | Mass: 16861.523 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#40: Protein | Mass: 14015.361 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#41: Protein | Mass: 14554.882 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#42: Protein | Mass: 11254.041 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#43: Protein | Mass: 12487.200 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#44: Protein | Mass: 13683.053 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#45: Protein | Mass: 12868.091 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#46: Protein | Mass: 11475.364 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#47: Protein | Mass: 10159.621 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#48: Protein | Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#49: Protein | Mass: 9263.946 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#50: Protein | Mass: 7734.896 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#51: Protein | Mass: 9421.018 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#52: Protein | Mass: 9577.268 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#53: Protein | Mass: 8392.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
-Non-polymers , 3 types, 440 molecules ![](data/chem/img/MG.gif)
![](data/chem/img/ZN.gif)
![](data/chem/img/HOH.gif)
![](data/chem/img/ZN.gif)
![](data/chem/img/HOH.gif)
#54: Chemical | ChemComp-MG / #55: Chemical | #56: Water | ChemComp-HOH / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: E. coli 70S d2d8 stapled ribosome / Type: RIBOSOME / Entity ID: #1-#53 / Source: NATURAL |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
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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 |
Image recording | Average exposure time: 1.8 sec. / Electron dose: 27 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) |
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Processing
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94371 / Algorithm: FOURIER SPACE / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5MDZ Accession code: 5MDZ / Source name: PDB / Type: experimental model |