Green-to-red　photoconvertible　fluorescent　proteins　(PCFPs)　serve　as　key　players　in　single-molecule　localization　super-resolution　imaging.　As　an　early　engineered　variant,　mEos3.2　has　limited　applications,　mostly　due　to　its　slow　maturation　rate.　The　recent　advent　of　a　novel　variant,　pcStar,　obtained　by　the　simple　mutation　of　only　three　amino　acids　(D28E/L93M/N166G)　in　mEos3.2,　exhibits　significantly　accelerated　maturation　and　enhanced　fluorescent　brightness.　This　improvement　represents　an　important　advance　in　the　field　of　biofluorescence　by　enabling　early　detection　with　reliable　signals,　essential　for　labelling　dynamic　biological　processes.　However,　the　mechanism　underlying　the　significant　improvement　in　fluorescent　performance　from　mEos3.2　to　pcStar　remains　elusive,　preventing　the　rational　design　of　more　robust　variants　through　mutagenesis.　In　this　study,　we　determined　the　crystal　structures　of　mEos3.2　and　pcStar　in　their　green　states　at　atomic　resolution　and　performed　molecular-dynamics　simulations　to　reveal　significant　divergences　between　the　two　proteins.　Our　structural　and　computational　analyses　revealed　crucial　features　that　are　distinctively　present　in　pcStar,　including　the　presence　of　an　extra　solvent　molecule,　high　conformational　stability　and　enhanced　interactions　of　the　chromophore　with　its　surroundings,　tighter　tertiary-structure　packing　and　dynamic　central-helical　deformation.　Resulting　from　the　triple　mutations,　all　of　these　structural　features　are　likely　to　establish　a　mechanistic　link　to　the　greatly　improved　fluorescent　performance　of　pcStar.　The　data　described　here　not　only　provide　a　good　example　illustrating　how　distant　amino-acid　substitutions　can　affect　the　structure　and　bioactivity　of　a　protein,　but　also　give　rise　to　strategic　considerations　for　the　future　engineering　of　more　widely　applicable　PCFPs.