+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-45573 | ||||||||||||||||||
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Title | 70S initiation complex (tRNA-fMet M1 + AUG start codon) | ||||||||||||||||||
Map data | Masked, sharpened map from homogeneous 3D refinement (sharpening B factor of -61.5 A^2), used for modeling | ||||||||||||||||||
Sample |
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Keywords | translation initiation / tRNA-fMet M1 / frameshifting / ribosome | ||||||||||||||||||
Function / homology | Function and homology information DnaA-L2 complex / negative regulation of DNA-templated DNA replication initiation / negative regulation of translational initiation / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / transcription antitermination / DNA-templated transcription termination / maintenance of translational fidelity ...DnaA-L2 complex / negative regulation of DNA-templated DNA replication initiation / negative regulation of translational initiation / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / transcription antitermination / 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 / ribosomal small subunit assembly / small ribosomal subunit / 5S rRNA binding / large ribosomal subunit rRNA binding / transferase activity / cytosolic small ribosomal subunit / ribosomal large subunit assembly / 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 / RNA binding / zinc ion binding / membrane / metal ion binding / cytosol / cytoplasm Similarity search - Function | ||||||||||||||||||
Biological species | Escherichia coli (E. coli) | ||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.75 Å | ||||||||||||||||||
Authors | Mattingly JM / Nguyen HA / Dunham CM | ||||||||||||||||||
Funding support | United States, 5 items
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Citation | Journal: J Biol Chem / Year: 2024 Title: Structural analysis of noncanonical translation initiation complexes. Authors: Jacob M Mattingly / Ha An Nguyen / Bappaditya Roy / Kurt Fredrick / Christine M Dunham / Abstract: Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a ...Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNA bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNA in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNA with C-G (called tRNA M1) reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNA M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNA variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNA and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNA anticodon stem by the ribosome can impact the start codon selection. | ||||||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_45573.map.gz | 230.3 MB | EMDB map data format | |
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Header (meta data) | emd-45573-v30.xml emd-45573.xml | 77.5 KB 77.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_45573_fsc.xml | 13.1 KB | Display | FSC data file |
Images | emd_45573.png | 224.5 KB | ||
Masks | emd_45573_msk_1.map | 244.1 MB | Mask map | |
Filedesc metadata | emd-45573.cif.gz | 15.2 KB | ||
Others | emd_45573_additional_1.map.gz emd_45573_half_map_1.map.gz emd_45573_half_map_2.map.gz | 183.8 MB 226.8 MB 226.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-45573 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-45573 | HTTPS FTP |
-Validation report
Summary document | emd_45573_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_45573_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_45573_validation.xml.gz | 22.3 KB | Display | |
Data in CIF | emd_45573_validation.cif.gz | 29 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-45573 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-45573 | HTTPS FTP |
-Related structure data
Related structure data | 9cg7MC 9ax7C 9ax8C 9cg5C 9cg6C C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_45573.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Masked, sharpened map from homogeneous 3D refinement (sharpening B factor of -61.5 A^2), used for modeling | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.045 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_45573_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : 70S initiation complex (tRNA-fMet M1 + AUG start codon)
+Supramolecule #1: 70S initiation complex (tRNA-fMet M1 + AUG start codon)
+Macromolecule #1: 50S ribosomal protein L33
+Macromolecule #2: 50S ribosomal protein L34
+Macromolecule #3: 50S ribosomal protein L35
+Macromolecule #4: 50S ribosomal protein L36
+Macromolecule #5: 50S ribosomal protein L31
+Macromolecule #7: 30S ribosomal protein S2
+Macromolecule #8: 30S ribosomal protein S3
+Macromolecule #9: 30S ribosomal protein S4
+Macromolecule #10: 30S ribosomal protein S5
+Macromolecule #11: 30S ribosomal protein S6
+Macromolecule #12: 30S ribosomal protein S7
+Macromolecule #13: 30S ribosomal protein S8
+Macromolecule #14: 30S ribosomal protein S9
+Macromolecule #15: 30S ribosomal protein S10
+Macromolecule #16: 30S ribosomal protein S11
+Macromolecule #17: 30S ribosomal protein S12
+Macromolecule #18: 30S ribosomal protein S13
+Macromolecule #19: 30S ribosomal protein S14
+Macromolecule #20: 30S ribosomal protein S15
+Macromolecule #21: 30S ribosomal protein S16
+Macromolecule #22: 30S ribosomal protein S17
+Macromolecule #23: 30S ribosomal protein S18
+Macromolecule #24: 30S ribosomal protein S19
+Macromolecule #25: 30S ribosomal protein S20
+Macromolecule #26: 30S ribosomal protein S21
+Macromolecule #31: 50S ribosomal protein L2
+Macromolecule #32: 50S ribosomal protein L3
+Macromolecule #33: 50S ribosomal protein L4
+Macromolecule #34: 50S ribosomal protein L5
+Macromolecule #35: 50S ribosomal protein L6
+Macromolecule #36: 50S ribosomal protein L9
+Macromolecule #37: 50S ribosomal protein L13
+Macromolecule #38: 50S ribosomal protein L14
+Macromolecule #39: 50S ribosomal protein L15
+Macromolecule #40: 50S ribosomal protein L16
+Macromolecule #41: 50S ribosomal protein L17
+Macromolecule #42: 50S ribosomal protein L18
+Macromolecule #43: 50S ribosomal protein L19
+Macromolecule #44: 50S ribosomal protein L20
+Macromolecule #45: 50S ribosomal protein L21
+Macromolecule #46: 50S ribosomal protein L22
+Macromolecule #47: 50S ribosomal protein L23
+Macromolecule #48: 50S ribosomal protein L24
+Macromolecule #49: 50S ribosomal protein L25
+Macromolecule #50: 50S ribosomal protein L27
+Macromolecule #51: 50S ribosomal protein L28
+Macromolecule #52: 50S ribosomal protein L29
+Macromolecule #53: 50S ribosomal protein L30
+Macromolecule #54: 50S ribosomal protein L32
+Macromolecule #6: 16S ribosomal RNA
+Macromolecule #27: mRNA
+Macromolecule #28: P-site tRNA-fMet M1
+Macromolecule #29: 23S ribosomal RNA
+Macromolecule #30: 5S ribosomal RNA
+Macromolecule #55: ZINC ION
+Macromolecule #56: MAGNESIUM ION
+Macromolecule #57: BETA-L-ASPARTIC ACID
-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: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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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: 2265 / Average electron dose: 58.4 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 79000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Details | Starting model was fit into the unsharpened 3D reconstruction in UCSF ChimeraX before performing real-space refinement with the sharpened map in PHENIX |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 64.3 |
Output model | PDB-9cg7: |