Visualizing the translation landscape in human cells at high resolution

Abstract

AbstractObtaining comprehensive structural descriptions of macromolecules within their natural cellular context holds immense potential for understanding fundamental biology and improving health. Here, we present the landscape of protein synthesis inside human cells in unprecedented detail obtained using an approach which combines automated cryo-focused ion beam (FIB) milling andin situsingle-particle cryo-electron microscopy (cryo-EM). With thisin situcryo-EM approach we resolved a 2.19 Å consensus structure of the human 80S ribosome and unveiled its 21 distinct functional states, nearly all higher than 3 Å resolution. In contrast toin vitrostudies, we identified protein factors, including SERBP1, EDF1 and NAC/3, not enriched on purified ribosomes. Most strikingly, we observed that SERBP1 binds to the ribosome in almost all translating and non-translating states to bridge the 60S and 40S ribosomal subunits. These newly observed binding sites suggest that SERBP1 may serve an important regulatory role in translation. We also uncovered a detailed interface between adjacent translating ribosomes which can form the helical polysome structure. Finally, we resolved high-resolution structures from cells treated with homoharringtonine and cycloheximide, and identified numerous polyamines bound to the ribosome, including a spermidine that interacts with cycloheximide bound at the E site of the ribosome, underscoring the importance of high-resolutionin situstudiesinthe complex native environment. Collectively, our work represents a significant advancement in detailed structural studies within cellular contexts.