1IOZ

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

Summary for 1IOZ

• Entry DOI: 10.2210/pdb1ioz/pdb
• Descriptor: TRANSFORMING PROTEIN P21/H-RAS-1, GUANOSINE-5'-DIPHOSPHATE (3 entities in total)
• Functional Keywords: ras, oncogene protein, gtp-binding protein, cell-free protein synthesis, riken structural genomics/proteomics initiative, rsgi, structural genomics, signaling protein
• Biological source: Homo sapiens (human)
• Cellular location: Cell membrane; Lipid-anchor; Cytoplasmic side: P01112
• Total number of polymer chains: 1
• Total formula weight: 19960.26

Authors

Kigawa, T.,Yamaguchi-Nunokawa, E.,Kodama, K.,Matsuda, T.,Yabuki, T.,RIKEN Structural Genomics/Proteomics Initiative (RSGI) (deposition date: 2001-04-18, release date: 2001-10-03, Last modification date: 2023-12-27)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (2 Å)