The article investigates the mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases (CRLs) in collaboration with E2 ubiquitin-conjugating enzymes. CRLs, in particular, use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains, which control a wide range of eukaryotic processes. The study uses cryogenic-electron microscopy (cryo-EM) to obtain structures that provide insights into how these chains are formed. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation by releasing the RING domain from the CRL without contacting UBE2R2. These findings suggest how poly-ubiquitylation can be accomplished by many E2s and E3s. The study further explores the molecular synergy between CRLs and UBE2R2, highlighting the role of the synergy loop in integrating donor and acceptor ubiquitins into the catalytic conformation. The results provide a structural basis for understanding the rapid and efficient formation of Lys48-linked poly-ubiquitin chains, which is crucial for timely protein degradation and various cellular processes.The article investigates the mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases (CRLs) in collaboration with E2 ubiquitin-conjugating enzymes. CRLs, in particular, use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains, which control a wide range of eukaryotic processes. The study uses cryogenic-electron microscopy (cryo-EM) to obtain structures that provide insights into how these chains are formed. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation by releasing the RING domain from the CRL without contacting UBE2R2. These findings suggest how poly-ubiquitylation can be accomplished by many E2s and E3s. The study further explores the molecular synergy between CRLs and UBE2R2, highlighting the role of the synergy loop in integrating donor and acceptor ubiquitins into the catalytic conformation. The results provide a structural basis for understanding the rapid and efficient formation of Lys48-linked poly-ubiquitin chains, which is crucial for timely protein degradation and various cellular processes.