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- EMDB-16059: Elongating E. coli 70S ribosome containing acylated tRNA(iMet) in... -
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Basic information
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Title | Elongating E. coli 70S ribosome containing acylated tRNA(iMet) in the P-site and Am6AA mRNA codon in the A-site after uncompleted di-peptide formation | |||||||||
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![]() | m6A / mRNA modification / tRNA recognition / TRANSLATION | |||||||||
Function / homology | ![]() 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 ...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 / 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 / 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 / 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: 2.21 Å | |||||||||
![]() | Koziej L / Glatt S | |||||||||
Funding support | European Union, 1 items
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![]() | ![]() Title: Modulation of translational decoding by mA modification of mRNA. Authors: Sakshi Jain / Lukasz Koziej / Panagiotis Poulis / Igor Kaczmarczyk / Monika Gaik / Michal Rawski / Namit Ranjan / Sebastian Glatt / Marina V Rodnina / ![]() ![]() Abstract: N-methyladenosine (mA) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. mA in the mRNA coding regions inhibits translation elongation. Here, we show how ...N-methyladenosine (mA) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. mA in the mRNA coding regions inhibits translation elongation. Here, we show how mA modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of mA fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. mA does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 452.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 88.5 KB 88.5 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 16.8 KB | Display | ![]() |
Images | ![]() | 159.3 KB | ||
Masks | ![]() | 512 MB | ![]() | |
Others | ![]() ![]() ![]() | 257.6 MB 474.7 MB 474.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8bhlMC ![]() 8bf7C ![]() 8bgeC ![]() 8bghC ![]() 8bh4C ![]() 8bhjC ![]() 8bhnC ![]() 8bhpC ![]() 8bilC ![]() 8bimC C: citing same article ( M: atomic model generated by this map |
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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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Voxel size | X=Y=Z: 0.86 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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Density Histograms |
-Additional map: Map combined from half maps used for generation...
File | emd_16059_additional_1.map | ||||||||||||
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Annotation | Map combined from half maps used for generation of the final high-resolution DeepEM enhanced map. | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_16059_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_16059_half_map_2.map | ||||||||||||
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Density Histograms |
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Sample components
+Entire : Elongating E. coli 70S ribosome containing acylated tRNA(iMet) in...
+Supramolecule #1: Elongating E. coli 70S ribosome containing acylated tRNA(iMet) in...
+Supramolecule #2: 50S subunit
+Supramolecule #3: 30S subunit
+Supramolecule #4: 23S and 5S rRNA
+Supramolecule #5: 16S rRNA
+Supramolecule #6: mRNA and tRNA
+Macromolecule #1: 50S ribosomal protein L2
+Macromolecule #2: 50S ribosomal protein L3
+Macromolecule #3: 50S ribosomal protein L4
+Macromolecule #4: 50S ribosomal protein L5
+Macromolecule #5: 50S ribosomal protein L6
+Macromolecule #6: 50S ribosomal protein L9
+Macromolecule #7: 50S ribosomal protein L13
+Macromolecule #8: 50S ribosomal protein L14
+Macromolecule #9: 50S ribosomal protein L15
+Macromolecule #10: 50S ribosomal protein L16
+Macromolecule #11: 50S ribosomal protein L17
+Macromolecule #12: 50S ribosomal protein L18
+Macromolecule #13: 50S ribosomal protein L19
+Macromolecule #14: 50S ribosomal protein L20
+Macromolecule #15: 50S ribosomal protein L21
+Macromolecule #16: 50S ribosomal protein L22
+Macromolecule #17: 50S ribosomal protein L23
+Macromolecule #18: 50S ribosomal protein L24
+Macromolecule #19: 50S ribosomal protein L25
+Macromolecule #20: 50S ribosomal protein L27
+Macromolecule #21: 50S ribosomal protein L28
+Macromolecule #22: 50S ribosomal protein L29
+Macromolecule #23: 50S ribosomal protein L30
+Macromolecule #24: 50S ribosomal protein L32
+Macromolecule #25: 50S ribosomal protein L33
+Macromolecule #26: 50S ribosomal protein L34
+Macromolecule #27: 50S ribosomal protein L35
+Macromolecule #28: 50S ribosomal protein L36
+Macromolecule #29: 30S ribosomal protein S2
+Macromolecule #30: 30S ribosomal protein S3
+Macromolecule #31: 30S ribosomal protein S4
+Macromolecule #32: 30S ribosomal protein S5
+Macromolecule #33: 30S ribosomal protein S6
+Macromolecule #34: 30S ribosomal protein S7
+Macromolecule #35: 30S ribosomal protein S8
+Macromolecule #36: 30S ribosomal protein S9
+Macromolecule #37: 30S ribosomal protein S10
+Macromolecule #38: 30S ribosomal protein S11
+Macromolecule #39: 30S ribosomal protein S12
+Macromolecule #40: 30S ribosomal protein S13
+Macromolecule #41: 30S ribosomal protein S14
+Macromolecule #42: 30S ribosomal protein S15
+Macromolecule #43: 30S ribosomal protein S16
+Macromolecule #44: 30S ribosomal protein S17
+Macromolecule #45: 30S ribosomal protein S18
+Macromolecule #46: 30S ribosomal protein S19
+Macromolecule #47: 30S ribosomal protein S20
+Macromolecule #48: 30S ribosomal protein S21
+Macromolecule #49: 23S rRNA RRLG-RRNA
+Macromolecule #50: 5S rRNA
+Macromolecule #51: 16S rRNA RRSB-RRNA
+Macromolecule #52: mRNA
+Macromolecule #53: Acylated P-site tRNA(fMet)
+Macromolecule #54: N-FORMYLMETHIONINE
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 10 mg/mL | |||||||||||||||
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Buffer | pH: 7.5 Component:
Details: 1x TAKM7 buffer | |||||||||||||||
Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV | |||||||||||||||
Details | Initiation complex containing: 1x TAKM7 buffer 1 mM DTT (dithiothreitol) 1 mM GTP (guanosine 5'-triphosphate) 2.0 uM 70S ribosomes 3.0 uM IF1, IF2, IF3 mix 6.0 uM A-site Am6AA mRNA 3.0 uM fMet-tRNAfMet Mixed 1:1 with a ternary complex containing: 1x TAKM7 buffer 3 mM PEP (phosphoenolpyruvate) 1 mM GTP (guanosine 5'-triphosphate) 1 mM DTT (dithiothreitol) 1% PK (pyruvate kinase) 0.02 uM EF-Ts 1.5 uM EF-Tu 0.3 uM Lys-tRNALys Incubated on ice for 60 seconds Final concentration of the sample: 20 A260 (absorbance at 260 nm, 10 mm path), 10 A280 (absorbance at 280 nm, 10 mm path) |
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Electron microscopy
Microscope | TFS KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 7172 / Average exposure time: 1.82 sec. / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.9 µm |
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
Details | Following rigid body fitting we used online Namdiantor tool to perform initial flexible fitting: https://namdinator.au.dk/namdinator/ |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | ![]() PDB-8bhl: |
-Atomic model buiding 2
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
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Output model | ![]() PDB-8bhl: |