The crystal structure of oxygen-evolving photosystem II (PSII) from Thermosynechococcus vulcanus was determined at 3.7-Å resolution. This structure provides detailed insights into the arrangement of subunits, cofactors, and the Mn cluster involved in water-splitting reactions. The structure was built using sequences of PSII large subunits (D1, D2, CP47, CP43), extrinsic proteins (33- and 12-kDa), cytochrome c550, and low molecular mass subunits. The 12-kDa protein, previously uncharacterized, was identified and shown to be directly connected to the Mn cluster. The structure revealed the arrangement of chlorophylls and cofactors, including two β-carotenes near the reaction center, which are important for secondary electron transfer pathways. The Mn cluster was found to be coordinated by D1-His-198 and D2-His-197, with the C terminus of D1 directly interacting with the Mn cluster. The structure also showed that the 12-kDa protein links the 33-kDa protein and cytochrome c550, suggesting a different organization of extrinsic proteins in cyanobacteria compared to higher plants. The arrangement of cofactors, including chl a, pheophytin, and β-carotenes, was analyzed, with the distances and orientations of these molecules providing insights into electron transfer pathways. The Mn cluster was found to be surrounded by a barrier formed by extrinsic proteins and subunits, protecting it from the bulk solution. The structure of PSII from T. vulcanus provides a more detailed understanding of the molecular interactions and cofactor arrangements in PSII, which is essential for elucidating the mechanism of water-splitting and oxygen evolution. This study contributes to the ongoing efforts to understand the function and regulation of PSII in photosynthesis.The crystal structure of oxygen-evolving photosystem II (PSII) from Thermosynechococcus vulcanus was determined at 3.7-Å resolution. This structure provides detailed insights into the arrangement of subunits, cofactors, and the Mn cluster involved in water-splitting reactions. The structure was built using sequences of PSII large subunits (D1, D2, CP47, CP43), extrinsic proteins (33- and 12-kDa), cytochrome c550, and low molecular mass subunits. The 12-kDa protein, previously uncharacterized, was identified and shown to be directly connected to the Mn cluster. The structure revealed the arrangement of chlorophylls and cofactors, including two β-carotenes near the reaction center, which are important for secondary electron transfer pathways. The Mn cluster was found to be coordinated by D1-His-198 and D2-His-197, with the C terminus of D1 directly interacting with the Mn cluster. The structure also showed that the 12-kDa protein links the 33-kDa protein and cytochrome c550, suggesting a different organization of extrinsic proteins in cyanobacteria compared to higher plants. The arrangement of cofactors, including chl a, pheophytin, and β-carotenes, was analyzed, with the distances and orientations of these molecules providing insights into electron transfer pathways. The Mn cluster was found to be surrounded by a barrier formed by extrinsic proteins and subunits, protecting it from the bulk solution. The structure of PSII from T. vulcanus provides a more detailed understanding of the molecular interactions and cofactor arrangements in PSII, which is essential for elucidating the mechanism of water-splitting and oxygen evolution. This study contributes to the ongoing efforts to understand the function and regulation of PSII in photosynthesis.