This study presents the cryoEM structure of the full-length HPV16 E6 protein in complex with E6AP and p53 at a resolution of 3.3 Å. The structure reveals extensive protein-protein interactions between HPV16 E6 and E6AP, explaining their picomolar binding affinity. These findings provide insights into the molecular basis of the ternary complex, which has been a target for therapeutic development in HPV-driven cancers. The structure shows that HPV16 E6 is sandwiched between E6AP and p53, forming a large interaction interface of approximately 2,361 Ų, contributing to the high-affinity binding. The study also identifies three key interaction sites between HPV16 E6 and E6AP, which are essential for p53 ubiquitination and complex formation. Rational mutations in HPV16 E6 were shown to disrupt the interaction with E6AP, reducing p53 ubiquitination and E6AP auto-ubiquitination. The results suggest that multiple interaction points are important for robust complex formation and subsequent ubiquitination. Additionally, the study found that HPV18 E6 exists in higher molecular weight species, indicating that it is fully occupied in complexes with E6AP. The findings highlight the importance of understanding the structural and mechanistic underpinnings of the HPV16 E6-E6AP-p53 complex for developing effective therapies against HPV-induced cancers.This study presents the cryoEM structure of the full-length HPV16 E6 protein in complex with E6AP and p53 at a resolution of 3.3 Å. The structure reveals extensive protein-protein interactions between HPV16 E6 and E6AP, explaining their picomolar binding affinity. These findings provide insights into the molecular basis of the ternary complex, which has been a target for therapeutic development in HPV-driven cancers. The structure shows that HPV16 E6 is sandwiched between E6AP and p53, forming a large interaction interface of approximately 2,361 Ų, contributing to the high-affinity binding. The study also identifies three key interaction sites between HPV16 E6 and E6AP, which are essential for p53 ubiquitination and complex formation. Rational mutations in HPV16 E6 were shown to disrupt the interaction with E6AP, reducing p53 ubiquitination and E6AP auto-ubiquitination. The results suggest that multiple interaction points are important for robust complex formation and subsequent ubiquitination. Additionally, the study found that HPV18 E6 exists in higher molecular weight species, indicating that it is fully occupied in complexes with E6AP. The findings highlight the importance of understanding the structural and mechanistic underpinnings of the HPV16 E6-E6AP-p53 complex for developing effective therapies against HPV-induced cancers.