In yeast, H3K4 methylation, the hallmark of active transcription, is known to be dependent on H2B ubiquitylation. We want to understand the mechanism of H2B mono-ubiquitylation by E3 ubiquitin ligase Bre1and E2 ubiquitin conjugating enzyme Rad6. Bre1 has a coiled coil motif (CCM) at the N-terminus and a RING finger domain at the C-terminal end. Previous studies have well confirmed the Interaction between Bre1-CCM and Rad6. This interaction is non-canonical E2-E3 interaction because most of the E2 enzyme binds to the RING-finger domain of E3. By co-expressing Bre1-CCM and Rad6 in E.coli, we purified the Bre1-CCM/Rad6 complex and further identified the X-ray crystal structure. To validate the structure obtained from X-ray crystallography, we further purified various mutant proteins and observed the interaction change between two proteins and also H2B ubiquitylation activity.