Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA. [Display omitted] •X-ray structures of major yeast eIF3 components and subcomplexes•Crosslinking coupled to mass-spectrometry analysis of 40S⋅eIF1⋅eIF3 complex•Integrative modeling reveals architecture of 40S⋅eIF1⋅eIF3 complex A hybrid approach drawing on X-ray structures, crosslinking coupled to mass spectrometry, electron microscopy, and integrative modeling yields mechanistic insights into how eIF3 coordinates translation initiation.