However, we failed to observe membrane morphology changes for the D151N mutant for a wide range of protein concentrations. Cryo-EM reconstructions and simulations of endophilin N-BAR revealed that oligomeric assembly on flat and tubular membranes is essential for inducing and stabilizing tubulation. Additionally, recent computer simulations have proposed that transient protein lattice formation/aggregation on flat membranes is Salvianolic-acid-A a prerequisite for tubulation induced by the N-BAR domain. Here, we used a chemical cross-linking assay to ask the question if failure in membrane budding by the D151N mutant was due to defective protein oligomerization. In the absence of crosslinkers, BIN1 N-BAR and variants were monomeric in solution under denaturing conditions. However, when liposomes were present,14α-hydroxy-Sprengerinin-C multiple oligomeric bands with molecular weight larger than a single dimer appeared on SDS-PAGE gels for the cases of WT and K35N, demonstrating that higher-ordered protein complexes were formed when associated with membranes. For these two proteins, after adding 5 mM BS3, a fraction of the crosslinked species became too large to be able to enter the resolving gel. Addition of BS3 crosslinker to the mixture of R154Q and liposomes resulted in diminishing dimer bands and an unresolvable pattern of oligomeric species on the gel. The unresolvable oligomer pattern of R154Q could be a result of the lowest membrane association affinity among all mutants. In contrast, we observed only dimer bands for D151N mutants in the presence of liposomes confirming that the capacity to form protein assemblies on the membrane is impaired for the D151N mutant. Even at high crosslinker concentration, absence of protein retention in the stacking gel indicated absence of D151N oligomers.