YK13 peptides assemble one-dimensionally into a fibril structure in the presence of ATP, but why did YK13-sfGFP adopt a droplet form with an isotropic spherical structure in its assembly? To answer this question, we examined ATP-dependent assembly in a test tube using purified YK13-sfGFP, which was expressed in E. coli (Fig. and Supplementary Fig. ). The results revealed that the r of YK13-sfGFP significantly decreased upon mixing with ATP, supporting its ATP-mediated self-assembly (Fig. ). Intriguingly, the mere addition of ATP to the YK13-sfGFP solution did not lead to droplets. DLS measurements indicated colloidal structures with a particle size of approximately 20 nm (Fig. ). TEM observation revealed spherical structures of 20 nm, which is consistent with the DLS results (Fig. ). Meanwhile, untagged sfGFP showed neither a decrease in r nor an increase in particle size (Fig. and Supplementary Fig. ). The CD spectrum of YK13-sfGFP altered upon addition of ATP; the difference spectrum for sfGFP displayed positive peaks at 210 nm and 235 nm, comparable to that of the YK13 peptide (Supplementary Fig. ). While YK13-sfGFP adopts a structure similar to that of YK13 peptide, it forms an oligomeric assembly, probably due to frustrated growth caused by steric hindrance of sfGFP. The r increased with increasing salt strength (Supplementary Fig. ), and YK13-sfGFP particle size decreased with increasing temperature (Supplementary Fig. ), suggesting that the electrostatic interaction contributed to assembly. Similar to ATP, pyrophosphate and GTP significantly reduced the r value (Fig. and Supplementary Fig. ). ADP displayed an increased concentration threshold for a response at concentrations exceeding 3 mM. In contrast, phosphate and AMP, with low anion valence, showed no changes even at 30 mM. CTP, UTP, poly-A (700-3500 kDa), and poly-glutamate (> 12 kDa) also exhibited a decrease in r, albeit less pronounced compared to the decrease observed with the addition of ATP or GTP. The complex with poly-A formed smaller colloids, presumably due to a significantly negative zeta potential, suppressing fusion through charge repulsion (Supplementary Fig. ).