Fig. 1: Cryo-EM sample preparation guided by single-molecule fluorescence data and overall architecture. | Nature Communications

Fig. 1: Cryo-EM sample preparation guided by single-molecule fluorescence data and overall architecture.

From: Structural basis for the transition from translation initiation to elongation by an 80S-eIF5B complex

Fig. 1

a Overview of translation initiation in eukaryotes. Two steps, regulated by GTPases eIF2 and eIF5B control the delivery of Met-tRNAiMet. b Cryo-EM samples were prepared and froze at the timepoint 40 s after mixing 48S PICs, eIF5B:GTP, and 60S. At this timepoint, ~40% of the total 80S particles are expected to be 80S EC, while the other ~60% are 80S IC with eIF5B-bound, as demonstrated by our previous single-molecule fluorescence data (Supplementary Fig. 1)11. c Overview of the final cryo-EM map obtained after maximum likelihood classifications in Relion44. Large subunit (60S) is colored in blue, small subunit (40S) yellow, Met-tRNAiMet green, mRNA gray, and eIF5B red. Bottom, details of the local map obtained for Met-tRNAiMet, mRNA, and eIF5B. Domain IV (DIV) of eIF5B is in close contact with the acceptor stem of Met-tRNAiMet. d Stereochemically refined models for Met-tRNAiMet, mRNA, and eIF5B with components indicated.

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