【Research Highlights】 We determined the three-dimensional structure at a high resolution of 2.0 Å of the giant protein complex "Photosystem II (PSII)," which decomposes water and generates oxygen during photosynthesis, after its three extrinsic proteins were removed and then reattached. It was found that the reattached three extrinsic proteins (PsbO, PsbV, and PsbU) returned to their correct positions on the main body of PSII. On the other hand, slight changes were observed in the orientation of bicarbonate ions involved in the oxygen evolution reaction and the arrangement of water molecules. Specifically, the arrangement of water molecules and the hydrogen bond network were disrupted in the "O1 channel," which is thought to be the pathway for water in the oxygen evolution reaction. This research revealed that even if the extrinsic proteins return to their correct positions, the oxygen evolution activity of photosynthesis may decrease because the arrangement of water molecules and small molecules inside PSII is not completely restored. 【Research Overview】 Associate Professor Ryo Nagao of the Faculty of Agriculture, Shizuoka University, along with Assistant Professor Yoshiki Nakajima and Professor Jian-Ren Shen of the Institute for Fundamental Chemistry (Interdisciplinary Field), Okayama University, determined the molecular structure of Photosystem II (PSII) (Note 1) from the thermophilic cyanobacterium Thermosynechococcus vulcanus after removing and reattaching the three extrinsic proteins PsbO, PsbV, and PsbU (Note 2), using X-ray crystallography (Note 3). The analysis revealed that the reattached PsbO, PsbV, and PsbU all bound to their correct positions on the main body of PSII. While the overall structure around the metal cluster involved in the oxygen evolution reaction was largely maintained, changes were observed in the orientation of bicarbonate ions and their interactions with surrounding water molecules. Furthermore, disruptions in the arrangement of water