1IOZ

Crystal Structure of the C-HA-RAS Protein Prepared by the Cell-Free Synthesis

1IOZ の概要

• エントリーDOI: 10.2210/pdb1ioz/pdb
• 分子名称: TRANSFORMING PROTEIN P21/H-RAS-1, GUANOSINE-5'-DIPHOSPHATE (3 entities in total)
• 機能のキーワード: ras, oncogene protein, gtp-binding protein, cell-free protein synthesis, riken structural genomics/proteomics initiative, rsgi, structural genomics, signaling protein
• 由来する生物種: Homo sapiens (human)
• 細胞内の位置: Cell membrane; Lipid-anchor; Cytoplasmic side: P01112
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 19960.26

構造登録者

Kigawa, T.,Yamaguchi-Nunokawa, E.,Kodama, K.,Matsuda, T.,Yabuki, T.,RIKEN Structural Genomics/Proteomics Initiative (RSGI) (登録日: 2001-04-18, 公開日: 2001-10-03, 最終更新日: 2023-12-27)

主引用文献

Kigawa, T.,Yamaguchi-Nunokawa, E.,Kodama, K.,Matsuda, T.,Yabuki, T.,Matsuda, N.,Ishitani, R.,Nureki, O.,Yokoyama, S.
Selenomethionine incorporation into a protein by cell-free synthesis
J.STRUCT.FUNCT.GENOM., 2:29-35, 2001

PubMed Abstract: Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.

PubMed: 12836672
DOI: 10.1023/A:1013203532303

実験手法

X-RAY DIFFRACTION (2 Å)