Subsequently, we investigated the molecular crowding effects on droplet formation of YK13-sfGFP, considering that various recombinant IDRs phase-separated in vitro under crowded conditions . The results revealed that a solution containing 5 µM YK13-sfGFP and 1 mM ATP exhibited highly spherical structures (roundness of 0.92 ± 0.08) under 3% PEG20k conditions, gradually growing in size over time (Fig. and Supplementary Fig. ). When varying the applied concentration, the partition coefficient remained almost constant, supporting the assertion that YK13-sfGFP exhibits characteristics of droplets (Supplementary Fig. ). The phase diagram showed that the phase boundary existed above 4 µM YK13-sfGFP and above 400 µM ATP (Supplementary Fig. ). We observed similar droplets upon the addition of ADP, pyrophosphate, and other NTPs, whereas smaller aggregates formed upon the addition of poly-A and poly-glutamate, carrying high valence anions (Supplementary Fig. ). Taking these findings together, YK13-sfGFP rapidly forms oligomers through interactions with poly-anion species driven by electrostatic interactions, followed by droplet formation in a crowded environment.