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Ribosomes catalyze protein synthesis by cycling through various functional states. These states have been extensively characterized in vitro, yet their distribution in actively translating human cells remains elusive. Here, we optimized a cryo-electron tomography-based approach and resolved ribosome structures inside human cells with a local resolution of up to 2.5 angstroms. These structures revealed the distribution of functional states of the elongation cycle, a Z tRNA binding site and the dynamics of ribosome expansion segments. In addition, we visualized structures of Homoharringtonine, a drug for chronic myeloid leukemia treatment, within the active site of the ribosome and found that its binding reshaped the landscape of translation. Overall, our work demonstrates that structural dynamics and drug effects can be assessed at near-atomic detail within human cells.One-Sentence SummarySnapshots of ribosome dynamics at near-atomic resolution within native and drug-treated human cells are revealed.

Zika virus (ZIKV) is an emergent flavivirus associated with severe neurological disorders. ZIKV NS3 protein is a viral protease that cleaves the ZIKV polyprotein precursor into individual viral proteins. In this study, we found that ZIKV NS3 by itself exhibited mitochondrial localization, which was quite different from its endoplasmic reticulum (ER) localization in ZIKV-infected cells. We screened viral proteins and identified NS2B as the bona fide recruiter of NS3 to the ER. The NS2B C-terminal tail interacted with NS3 protease domain to retain NS3 on the ER. β-Sheet motifs that formed between NS2B and the NS3 protease domain played important roles in their interaction, while mutation in the β-strand of NS2B attenuated NS2B-NS3 interaction and impaired the ability of NS3 protease to cleave the polyprotein precursor into multiple viral proteins. Consequently, NS2B mutations led to severe inhibition of ZIKV replication and production due to insufficient NS3 protease activity. In summary, our study reveals the critical role of NS2B in NS3 recruitment and protease function and provides mechanistic insight into ZIKV replication.