Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1. This study reveals the mechanism of a naturally occurring E3-independent ubiquitination reaction catalyzed by the human E2 enzyme UBE2E1. By solving the structure of UBE2E1 in complex with a SETDB1-derived peptide, the researchers developed an E3-free enzymatic strategy called SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitinated sites, ubiquitin chain linkages, and lengths. This strategy can also be used to generate site-specific branched ubiquitin chains or even NEDD8-modified proteins. The work deepens the understanding of how E3-free substrate ubiquitination occurs in human cells and provides a practical approach for obtaining ubiquitinated proteins to dissect the biochemical functions of ubiquitination. The study demonstrates that UBE2E1 specifically recognizes and ubiquitinates the hexapeptide KEGYES in an "L" shape, localizing it near its active site for ubiquitin transfer in an E3-free mechanism. The SUE1 strategy enables the generation of ubiquitinated proteins with defined sites, ubiquitin chain linkages, and lengths, and can be combined with other enzymatic methods to achieve different forms of ubiquitination at different sites of the same protein. Additionally, the SUE1 strategy is applicable to ubiquitin-like NEDD8 modification, offering an unprecedented general enzymatic strategy for introducing NEDD8 modification into given proteins. The study also shows that the K29/48-branched Ub chain is less efficient in triggering proteasomal degradation compared to the K48-linked Ub chain. The results highlight the role of ubiquitin-related enzymes as an enzymatic tool library for generating ubiquitinated substrates with native isopeptide bonds.Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1. This study reveals the mechanism of a naturally occurring E3-independent ubiquitination reaction catalyzed by the human E2 enzyme UBE2E1. By solving the structure of UBE2E1 in complex with a SETDB1-derived peptide, the researchers developed an E3-free enzymatic strategy called SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitinated sites, ubiquitin chain linkages, and lengths. This strategy can also be used to generate site-specific branched ubiquitin chains or even NEDD8-modified proteins. The work deepens the understanding of how E3-free substrate ubiquitination occurs in human cells and provides a practical approach for obtaining ubiquitinated proteins to dissect the biochemical functions of ubiquitination. The study demonstrates that UBE2E1 specifically recognizes and ubiquitinates the hexapeptide KEGYES in an "L" shape, localizing it near its active site for ubiquitin transfer in an E3-free mechanism. The SUE1 strategy enables the generation of ubiquitinated proteins with defined sites, ubiquitin chain linkages, and lengths, and can be combined with other enzymatic methods to achieve different forms of ubiquitination at different sites of the same protein. Additionally, the SUE1 strategy is applicable to ubiquitin-like NEDD8 modification, offering an unprecedented general enzymatic strategy for introducing NEDD8 modification into given proteins. The study also shows that the K29/48-branched Ub chain is less efficient in triggering proteasomal degradation compared to the K48-linked Ub chain. The results highlight the role of ubiquitin-related enzymes as an enzymatic tool library for generating ubiquitinated substrates with native isopeptide bonds.