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Yorodumi- EMDB-13017: RqcH DR variant bound to 50S-peptidyl-tRNA-RqcP RQC complex (rigi... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13017 | ||||||||||||||||||||||||
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Title | RqcH DR variant bound to 50S-peptidyl-tRNA-RqcP RQC complex (rigid body refinement) | ||||||||||||||||||||||||
Map data | Bacillus subtilis ribosomal large subunit in complex with peptidyl-tRNA, RqcP, and variant D97A/R98A RqcH. From RELION Refine3D, low-pass filtered to 5A. | ||||||||||||||||||||||||
Sample |
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Function / homology | Function and homology information positive regulation of rRNA processing / nucleoid / ribosomal large subunit binding / rescue of stalled ribosome / rRNA processing / large ribosomal subunit / ribosomal large subunit assembly / transferase activity / large ribosomal subunit rRNA binding / 5S rRNA binding ...positive regulation of rRNA processing / nucleoid / ribosomal large subunit binding / rescue of stalled ribosome / rRNA processing / large ribosomal subunit / ribosomal large subunit assembly / transferase activity / large ribosomal subunit rRNA binding / 5S rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / response to antibiotic / mRNA binding / DNA binding / RNA binding / cytoplasm Similarity search - Function | ||||||||||||||||||||||||
Biological species | Bacillus subtilis subsp. subtilis str. 168 (bacteria) | ||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||||||||||||||||||||
Authors | Crowe-McAuliffe C / Wilson DN | ||||||||||||||||||||||||
Funding support | Sweden, Germany, Estonia, 7 items
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Citation | Journal: Nucleic Acids Res / Year: 2021 Title: RqcH and RqcP catalyze processive poly-alanine synthesis in a reconstituted ribosome-associated quality control system. Authors: Hiraku Takada / Caillan Crowe-McAuliffe / Christine Polte / Zhanna Yu Sidorova / Victoriia Murina / Gemma C Atkinson / Andrey L Konevega / Zoya Ignatova / Daniel N Wilson / Vasili Hauryliuk / Abstract: In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the ...In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway. | ||||||||||||||||||||||||
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_13017.map.gz | 260 MB | EMDB map data format | |
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Header (meta data) | emd-13017-v30.xml emd-13017.xml | 51.3 KB 51.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13017_fsc.xml | 14.9 KB | Display | FSC data file |
Images | emd_13017.png | 113.1 KB | ||
Masks | emd_13017_msk_1.map | 282.6 MB | Mask map | |
Others | emd_13017_additional_1.map.gz emd_13017_half_map_1.map.gz emd_13017_half_map_2.map.gz | 225.2 MB 225.4 MB 225.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13017 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13017 | HTTPS FTP |
-Validation report
Summary document | emd_13017_validation.pdf.gz | 527 KB | Display | EMDB validaton report |
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Full document | emd_13017_full_validation.pdf.gz | 526.1 KB | Display | |
Data in XML | emd_13017_validation.xml.gz | 22.6 KB | Display | |
Data in CIF | emd_13017_validation.cif.gz | 29 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13017 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13017 | HTTPS FTP |
-Related structure data
Related structure data | 7opeMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10726 (Title: Affinity-purified RqcH-DR-ribosome-associated quality control complexes from Bacillus subtilis Data size: 213.5 Data #1: Unaligned multiframe micrographs of RqcH-DR-mutant immunoprecipitation [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_13017.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Bacillus subtilis ribosomal large subunit in complex with peptidyl-tRNA, RqcP, and variant D97A/R98A RqcH. From RELION Refine3D, low-pass filtered to 5A. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.82 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_13017_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Bacillus subtilis ribosomal large subunit in complex with...
File | emd_13017_additional_1.map | ||||||||||||
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Annotation | Bacillus subtilis ribosomal large subunit in complex with peptidyl-tRNA, RqcP, and variant D97A/R98A RqcH. From RELION Refine3D. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map from RELION Refine3D.
File | emd_13017_half_map_1.map | ||||||||||||
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Annotation | Half map from RELION Refine3D. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map from RELION Refine3D.
File | emd_13017_half_map_2.map | ||||||||||||
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Annotation | Half map from RELION Refine3D. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : RQC complex consisting of 50S large ribosomal subunit, peptidyl-t...
+Supramolecule #1: RQC complex consisting of 50S large ribosomal subunit, peptidyl-t...
+Macromolecule #1: 23S rRNA
+Macromolecule #2: 5S rRNA
+Macromolecule #22: tRNA-Ala-UGC
+Macromolecule #3: 50S ribosomal protein L2
+Macromolecule #4: 50S ribosomal protein L3
+Macromolecule #5: 50S ribosomal protein L4
+Macromolecule #6: 50S ribosomal protein L5
+Macromolecule #7: 50S ribosomal protein L6
+Macromolecule #8: 50S ribosomal protein L11
+Macromolecule #9: 50S ribosomal protein L10
+Macromolecule #10: 50S ribosomal protein L13
+Macromolecule #11: 50S ribosomal protein L14
+Macromolecule #12: 50S ribosomal protein L15
+Macromolecule #13: 50S ribosomal protein L16
+Macromolecule #14: 50S ribosomal protein L17
+Macromolecule #15: 50S ribosomal protein L18
+Macromolecule #16: 50S ribosomal protein L19
+Macromolecule #17: 50S ribosomal protein L20
+Macromolecule #18: 50S ribosomal protein L21
+Macromolecule #19: 50S ribosomal protein L22
+Macromolecule #20: 50S ribosomal protein L23
+Macromolecule #21: 50S ribosomal protein L24
+Macromolecule #23: 50S ribosomal protein L27
+Macromolecule #24: 50S ribosomal protein L28
+Macromolecule #25: 50S ribosomal protein L29
+Macromolecule #26: 50S ribosomal protein L30
+Macromolecule #27: 50S ribosomal protein L32
+Macromolecule #28: 50S ribosomal protein L33 1
+Macromolecule #29: 50S ribosomal protein L34
+Macromolecule #30: 50S ribosomal protein L35
+Macromolecule #31: 50S ribosomal protein L36
+Macromolecule #32: Uncharacterized protein YabO
+Macromolecule #33: Rqc2 homolog RqcH
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Number real images: 2510 / Average exposure time: 3.0 sec. / Average electron dose: 34.8 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |