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Yorodumi- EMDB-12694: Cryo-EM structure of an Escherichia coli TnaC(R23F)-ribosome comp... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-12694 | |||||||||||||||
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Title | Cryo-EM structure of an Escherichia coli TnaC(R23F)-ribosome complex stalled in response to L-tryptophan | |||||||||||||||
Map data | E.coli 70S ribosome stalled on TnaC-(R23F) with Pro24 in the P-site. | |||||||||||||||
Sample |
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Keywords | ribosome / regulation / TnaC / arrest peptide / L-tryptophan / translation / indole / stalling | |||||||||||||||
Function / homology | Function and homology information negative regulation of cytoplasmic translational initiation / 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 translational initiation / 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 / 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 / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / cytosolic ribosome assembly / transcription elongation factor complex / regulation of DNA-templated transcription elongation / DNA endonuclease activity / response to reactive oxygen species / transcription antitermination / regulation of cell growth / translational initiation / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit assembly / large ribosomal subunit rRNA binding / 5S rRNA binding / transferase activity / small ribosomal subunit / cytosolic small ribosomal subunit / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / molecular adaptor activity / negative regulation of translation / rRNA binding / 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 / cytosol / cytoplasm Similarity search - Function | |||||||||||||||
Biological species | Escherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.4 Å | |||||||||||||||
Authors | van der Stel AX / Gordon ER | |||||||||||||||
Funding support | France, United States, 4 items
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Citation | Journal: Nat Commun / Year: 2021 Title: Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling. Authors: Anne-Xander van der Stel / Emily R Gordon / Arnab Sengupta / Allyson K Martínez / Dorota Klepacki / Thomas N Perry / Alba Herrero Del Valle / Nora Vázquez-Laslop / Matthew S Sachs / Luis R ...Authors: Anne-Xander van der Stel / Emily R Gordon / Arnab Sengupta / Allyson K Martínez / Dorota Klepacki / Thomas N Perry / Alba Herrero Del Valle / Nora Vázquez-Laslop / Matthew S Sachs / Luis R Cruz-Vera / C Axel Innis / Abstract: Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive ...Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite. | |||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12694.map.gz | 120.4 MB | EMDB map data format | |
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Header (meta data) | emd-12694-v30.xml emd-12694.xml | 87.5 KB 87.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_12694_fsc.xml | 16.4 KB | Display | FSC data file |
Images | emd_12694.png | 240 KB | ||
Masks | emd_12694_msk_1.map | 131 MB | Mask map | |
Filedesc metadata | emd-12694.cif.gz | 15.5 KB | ||
Others | emd_12694_half_map_1.map.gz emd_12694_half_map_2.map.gz | 306 MB 306 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12694 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12694 | HTTPS FTP |
-Validation report
Summary document | emd_12694_validation.pdf.gz | 1.2 MB | Display | EMDB validaton report |
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Full document | emd_12694_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | emd_12694_validation.xml.gz | 24.6 KB | Display | |
Data in CIF | emd_12694_validation.cif.gz | 31.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12694 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12694 | HTTPS FTP |
-Related structure data
Related structure data | 7o1aMC 7o19C 7o1cC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10695 (Title: Cryo-EM structure of an Escherichia coli TnaC-ribosome complex stalled in response to L-tryptophan Data size: 6.9 TB Data #1: Raw multiframe micrographs of TnaC-WT-70S ribosome stalled on L-Trp [micrographs - multiframe] Data #2: Raw multiframe micrographs of TnaC-R23F-70S ribosome stalled on L-Trp (dataset B) [micrographs - multiframe] Data #3: Raw multiframe micrographs of TnaC-R23F-70S ribosome stalled on L-Trp (dataset A) [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_12694.map.gz / Format: CCP4 / Size: 131 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | E.coli 70S ribosome stalled on TnaC-(R23F) with Pro24 in the P-site. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.835 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_12694_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: Halfmap 1 of E.coli 70S ribosome stalled on TnaC-(R23F)...
File | emd_12694_half_map_1.map | ||||||||||||
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Annotation | Halfmap_1 of E.coli 70S ribosome stalled on TnaC-(R23F) with Pro24 in the P-site. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Halfmap 2 of E.coli 70S ribosome stalled on TnaC-(R23F)...
File | emd_12694_half_map_2.map | ||||||||||||
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Annotation | Halfmap_2 of E.coli 70S ribosome stalled on TnaC-(R23F) with Pro24 in the P-site. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : 70S ribosome stalled during translation of TnaC(R23F) with Pro24 ...
+Supramolecule #1: 70S ribosome stalled during translation of TnaC(R23F) with Pro24 ...
+Supramolecule #2: 30S subunit
+Supramolecule #3: 50S subunit
+Supramolecule #4: P-site tRNA-Pro - TnaC-(R23F) nascent chain
+Supramolecule #5: mRNA
+Macromolecule #1: Ribosomal RNA 16S
+Macromolecule #22: Ribosomal RNA 23S
+Macromolecule #23: Ribosomal RNA 5S
+Macromolecule #54: mRNA
+Macromolecule #55: P-site tRNA-Pro
+Macromolecule #2: 30S ribosomal protein S2
+Macromolecule #3: 30S ribosomal protein S3
+Macromolecule #4: 30S ribosomal protein S4
+Macromolecule #5: 30S ribosomal protein S5
+Macromolecule #6: 30S ribosomal protein S6
+Macromolecule #7: 30S ribosomal protein S7
+Macromolecule #8: 30S ribosomal protein S8
+Macromolecule #9: 30S ribosomal protein S9
+Macromolecule #10: 30S ribosomal protein S10
+Macromolecule #11: 30S ribosomal protein S11
+Macromolecule #12: 30S ribosomal protein S12
+Macromolecule #13: 30S ribosomal protein S13
+Macromolecule #14: 30S ribosomal protein S14
+Macromolecule #15: 30S ribosomal protein S15
+Macromolecule #16: 30S ribosomal protein S16
+Macromolecule #17: 30S ribosomal protein S17
+Macromolecule #18: 30S ribosomal protein S18
+Macromolecule #19: 30S ribosomal protein S19
+Macromolecule #20: 30S ribosomal protein S20
+Macromolecule #21: 30S ribosomal protein S21
+Macromolecule #24: 50S ribosomal protein L2
+Macromolecule #25: 50S ribosomal protein L3
+Macromolecule #26: 50S ribosomal protein L4
+Macromolecule #27: 50S ribosomal protein L5
+Macromolecule #28: 50S ribosomal protein L6
+Macromolecule #29: 50S ribosomal protein L9
+Macromolecule #30: 50S ribosomal protein L31
+Macromolecule #31: 50S ribosomal protein L13
+Macromolecule #32: 50S ribosomal protein L14
+Macromolecule #33: 50S ribosomal protein L15
+Macromolecule #34: 50S ribosomal protein L16
+Macromolecule #35: 50S ribosomal protein L17
+Macromolecule #36: 50S ribosomal protein L18
+Macromolecule #37: 50S ribosomal protein L19
+Macromolecule #38: 50S ribosomal protein L20
+Macromolecule #39: 50S ribosomal protein L21
+Macromolecule #40: 50S ribosomal protein L22
+Macromolecule #41: 50S ribosomal protein L23
+Macromolecule #42: 50S ribosomal protein L24
+Macromolecule #43: 50S ribosomal protein L25
+Macromolecule #44: 50S ribosomal protein L27
+Macromolecule #45: 50S ribosomal protein L28
+Macromolecule #46: 50S ribosomal protein L29
+Macromolecule #47: 50S ribosomal protein L30
+Macromolecule #48: 50S ribosomal protein L32
+Macromolecule #49: 50S ribosomal protein L33
+Macromolecule #50: 50S ribosomal protein L34
+Macromolecule #51: 50S ribosomal protein L35
+Macromolecule #52: 50S ribosomal protein L36
+Macromolecule #53: TnaC - Tryptophanase leader peptide - R23F
+Macromolecule #56: MAGNESIUM ION
+Macromolecule #57: POTASSIUM ION
+Macromolecule #58: ZINC ION
+Macromolecule #59: TRYPTOPHAN
+Macromolecule #60: water
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL | |||||||||||||||
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Buffer | pH: 7.5 Component:
Details: All buffers used during the preparation contained 2 mM L-tryptophan. | |||||||||||||||
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.025 kPa | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV | |||||||||||||||
Details | Sample was purified using sucrose gradient ultracentrifugation, diluted to 200 nM and applied to grids. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 8270 / Average electron dose: 44.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 59880 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -1.6 µm / Nominal defocus min: -0.4 µm / Nominal magnification: 165000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |