|Entry||Database: EMDB / ID: EMD-4331|
|Title||Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map B)|
|Sample||Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (model B)|
|Biological species||Kluyveromyces lactis (yeast)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.5 Å|
|Authors||Llacer JL / Hussain T / Gordiyenko Y / Ramakrishnan V|
|Citation||Journal: Elife / Year: 2018|
Title: Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition.
Authors: Jose Luis Llácer / Tanweer Hussain / Adesh K Saini / Jagpreet Singh Nanda / Sukhvir Kaur / Yuliya Gordiyenko / Rakesh Kumar / Alan G Hinnebusch / Jon R Lorsch / V Ramakrishnan /
Abstract: In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNA in a 'P' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) ...In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNA in a 'P' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNA. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNA interaction influenced initiation at near-cognate UUG codons and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.
|Validation Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
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|File||Download / File: emd_4331.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.34 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Half map: #1
|Projections & Slices|
-Half map: #2
-Entire Structure of a partial yeast 48S preinitiation complex with eIF5 ...
|Entire||Name: Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (model B)|
Number of components: 1
-Component #1: protein, Structure of a partial yeast 48S preinitiation complex w...
|Protein||Name: Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (model B)|
Recombinant expression: No
|Mass||Theoretical: 1.8 MDa|
|Source||Species: Kluyveromyces lactis (yeast)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.15 mg/mL / pH: 6.5|
|Vitrification||Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %|
-Electron microscopy imaging
Model: Tecnai Polara / Image courtesy: FEI Company
|Imaging||Microscope: FEI POLARA 300|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 78000.0 X (nominal), 104478.0 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 3500.0 nm|
|Specimen Holder||Model: OTHER / Temperature: (90.0 - 100.0 K)|
|Camera||Detector: FEI FALCON III (4k x 4k)|
|Image acquisition||Number of digital images: 3600 |
Details: Images were collected in movie-mode at 32 frames per second
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 54699 / Details: FEI Falcon III|
|3D reconstruction||Algorithm: FOURIER SPACE / Software: RELION / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF|
-Atomic model buiding
|Modeling #1||Target criteria: FSC / Refinement space: RECIPROCAL / Overall bvalue: 54|
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