This article investigates the mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases (CRLs). Using cryogenic-electron microscopy (cryo-EM), the study reveals the structural basis for how CRLs, in collaboration with E2 ubiquitin-conjugating enzymes, form these chains. The CRL RING domain activates the E2-bound ubiquitin and shapes a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The study also shows how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation by releasing the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s. The enzymatic assembly of poly-ubiquitin chains onto protein substrates is a defining feature of eukaryotic cell biology. Ubiquitin chain formation determines the fates of substrates, such as affecting their cellular localization or triggering degradation by the 26S proteasome. Poly-ubiquitin chains are forged during the covalent joining of a ubiquitin bound to a protein substrate with an enzyme-activated ubiquitin. Ubiquitin is a highly conserved protein containing seven lysine residues that may serve as linkage points for poly-ubiquitin chains. Lys48 is of particular importance as it is the only ubiquitin lysine residue required for viability in yeast and has been identified as the most abundant poly-ubiquitin chain linkage type in cells. Cdc34/UBE2R-family E2s are specialized in forging ubiquitin chains with Lys48-linkages that elicit 26S proteasome-dependent protein degradation. CRLs and Cdc34/UBE2R-catalyzed poly-ubiquitylation control gene expression, metabolism, signaling, protein trafficking, targeted protein degradation, and more. Understanding how CRLs, together with UBE2R-type E2s, poly-ubiquitylate substrates is important. CRLs are a large family of modular multi-subunit complexes, with approximately 300 members in humans. CRLs recruit substrate 'degron' motifs that bind to a specific substrate receptor module. Numerous distinct substrate receptors bind interchangeably to core cullin-RING modules. For example, F-box proteins are substrate receptors that function with the cullin-RING module CUL1–RBX1. BC-box proteins are substrate receptors that function with CUL2–RBX1. CRLs must partner with ubiquitin-carrying enzymes, which typically specialize in either 'priming' or 'extending' ubiquitin chains. CThis article investigates the mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases (CRLs). Using cryogenic-electron microscopy (cryo-EM), the study reveals the structural basis for how CRLs, in collaboration with E2 ubiquitin-conjugating enzymes, form these chains. The CRL RING domain activates the E2-bound ubiquitin and shapes a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The study also shows how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation by releasing the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s. The enzymatic assembly of poly-ubiquitin chains onto protein substrates is a defining feature of eukaryotic cell biology. Ubiquitin chain formation determines the fates of substrates, such as affecting their cellular localization or triggering degradation by the 26S proteasome. Poly-ubiquitin chains are forged during the covalent joining of a ubiquitin bound to a protein substrate with an enzyme-activated ubiquitin. Ubiquitin is a highly conserved protein containing seven lysine residues that may serve as linkage points for poly-ubiquitin chains. Lys48 is of particular importance as it is the only ubiquitin lysine residue required for viability in yeast and has been identified as the most abundant poly-ubiquitin chain linkage type in cells. Cdc34/UBE2R-family E2s are specialized in forging ubiquitin chains with Lys48-linkages that elicit 26S proteasome-dependent protein degradation. CRLs and Cdc34/UBE2R-catalyzed poly-ubiquitylation control gene expression, metabolism, signaling, protein trafficking, targeted protein degradation, and more. Understanding how CRLs, together with UBE2R-type E2s, poly-ubiquitylate substrates is important. CRLs are a large family of modular multi-subunit complexes, with approximately 300 members in humans. CRLs recruit substrate 'degron' motifs that bind to a specific substrate receptor module. Numerous distinct substrate receptors bind interchangeably to core cullin-RING modules. For example, F-box proteins are substrate receptors that function with the cullin-RING module CUL1–RBX1. BC-box proteins are substrate receptors that function with CUL2–RBX1. CRLs must partner with ubiquitin-carrying enzymes, which typically specialize in either 'priming' or 'extending' ubiquitin chains. C