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Two-dimensional crystallization and cryo-electron microscopy of photosystem II
K Nakazato1, C Toyoshima, I Enami
1Presto, JRDC, Wako Saitama, Japan.
Journal of Molecular Biology
|March 29, 1996
Summary
Researchers crystallized spinach photosystem II (PSII) into 2D sheets and vesicles for cryo-electron microscopy. This structural analysis revealed the arrangement and subunits of PSII particles, aiding in understanding its function.
Area of Science:
- Biophysics
- Structural Biology
- Photosynthesis Research
Background:
- Photosystem II (PSII) is crucial for oxygenic photosynthesis.
- Obtaining well-ordered PSII crystals is essential for high-resolution structural studies.
- Previous studies have faced challenges in crystallizing membrane proteins like PSII.
Purpose of the Study:
- To develop and characterize two-dimensional (2D) crystals of spinach Photosystem II (PSII).
- To determine the unit cell dimensions, packing arrangement, and constituent subunits of the PSII particles within the 2D crystals.
- To generate a projection map of PSII at 20 Å resolution to understand its structural organization.
Main Methods:
- Conventional detergent dialysis using n-heptyl-beta-D-thioglucoside to solubilize spinach PSII monomers.
- Formation of 2D crystals in the form of cylindrical vesicles or monolayer sheets.
- Cryo-electron microscopy with low-dose imaging of unstained crystals embedded in ice.
- Digital image processing for analyzing crystal images.
- SDS-PAGE and immunoblot analysis to identify constituent subunits.
Main Results:
- Successfully obtained 2D crystals of spinach PSII suitable for cryo-electron microscopy.
- Identified the plane group as p22(1)2(1) with a rectangular unit cell (16.7 nm x 15.3 nm) containing four PSII monomers.
- Confirmed the presence of CP47, D1, D2, cytochrome b-559, and psbI proteins within the 2D crystals.
- Generated a 20 Å resolution projection map revealing an asymmetrical monomer shape (8.1 nm x 7.5 nm) with a central high-density core assigned to the D1/D2 heterodimer.
Conclusions:
- The developed method yields high-quality 2D crystals of spinach PSII, enabling detailed structural analysis.
- The structural data provides insights into the arrangement of PSII subunits, particularly the D1/D2 heterodimer core.
- These findings contribute to a better understanding of the structure and function of Photosystem II in photosynthesis.