|Entry||Database: EMDB / ID: 2604|
|Title||Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES in a rotated stated with masked 40S|
|Map data||Map generated through masking the 40S subunit from the EMD-12385 map and performing a re-classification followed by re-refinement both process in the presence of the mask.|
|Sample||Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with 40S masked and re-refined:|
ribosome-eukaryote / CrPV-IRES
|Keywords||translation / ribosomes / eukaryote / initiation|
|Source||Kluyveromyces lactis (Kluyveromyces lactis) / Cricket paralysis virus|
|Method||single particle reconstruction / cryo EM / 3.8 Å resolution|
|Authors||Fernandez IS / Bai X / Scheres SHW / Ramakrishnan V|
|Citation||Journal: Cell / Year: 2014|
Title: Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome.
Authors: Israel S Fernández / Xiao-Chen Bai / Garib Murshudov / Sjors H W Scheres / V Ramakrishnan
Abstract: The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By ...The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively. In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit. The structure and accompanying factor-binding data show that CrPV-IRES binding mimics a pretranslocation rather than initiation state of the ribosome. Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.
|Validation Report||PDB-ID: 4v92|
SummaryFull reportAbout validation report
|Date||Deposition: Mar 5, 2014 / Header (metadata) release: Apr 9, 2014 / Map release: May 21, 2014 / Last update: Jun 11, 2014|
|Structure viewer||EM map: |
Downloads & links
|File||emd_2604.map.gz (map file in CCP4 format, 128001 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.34 Å|
CCP4 map header:
-Entire Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with ...
|Entire||Name: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with 40S masked and re-refined|
Number of components: 2
-Component #1: ribosome-eukaryote, Kluyveromyces lactis 40S ribosome
|Ribosome-eukaryote||Name: Kluyveromyces lactis 40S ribosome / Eukaryote: SSU 40S, SSU RNA 18S / Recombinant expression: No|
|Source||Species: Kluyveromyces lactis (Kluyveromyces lactis)|
-Component #2: protein, CrPV-IRES
|Protein||Name: CrPV-IRES / Recombinant expression: No|
|Source||Species: Cricket paralysis virus|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.1 mg/ml|
Buffer solution: MES-KOH, 40mM K-acetate, 10mM NH4-acetate, 8mM Mg-acetate, 2mM DTT
|Support film||400mesh Cu, Quantifoil R2/2|
|Vitrification||Instrument: FEI VITROBOT MARK I / Cryogen name: PROPANE / Temperature: 120 K / Humidity: 90 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS / Date: Jul 7, 2013|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: OTHER|
|Lens||Magnification: 47000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1.8 - 3 nm|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: FEI FALCON II (4k x 4k)|
|Image acquisition||Number of digital images: 1900|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 25969|
Details: Masked 40S area and focused classification and refined in the presence of the mask
|3D reconstruction||Algorithm: Relion / Software: Relion / CTF correction: Each particle / Resolution: 3.8 Å / Resolution method: FSC 0.143, gold-standard|
-Atomic model buiding
|Modeling #1||Software: Chimera, refmac / Refinement protocol: flexible / Target criteria: R-factor, FSC / Refinement space: RECIPROCAL|
Input PDB model: 3U5B
Overall bvalue: 60
|Modeling #2||Software: Chimera, refmac / Refinement protocol: flexible / Target criteria: R-factor, FSC / Refinement space: RECIPROCAL|
Input PDB model: 3U5C
Overall bvalue: 60
|Modeling #3||Software: Chimera, refmac / Refinement protocol: flexible / Target criteria: R-factor, FSC / Refinement space: RECIPROCAL|
Input PDB model: 3U5D
Overall bvalue: 60
|Modeling #4||Software: Chimera, refmac / Refinement protocol: flexible / Target criteria: R-factor, FSC / Refinement space: RECIPROCAL|
Input PDB model: 3U5E
Overall bvalue: 60
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