1 Calmodulin • Conformation? • Dynamics? Papers 1. Wilson & Brunger (2000), J. Mol. Biol. 301:1237-1256. PMID: 10966818 2. Meador, Means & Quiocho (1992), Science 257:1251-1255. PMID: 1519061* 3. Fallon & Quiocho (2003), Structure 11:1303-1307. PMID: 14527397 4. Horvath et al. (2005), J. Biol. Chem. 280:8266-8274. PMID: 15596444 5. Drum et al. (2002), Nature 415:396-402. PMID: 11807546 6. Drum et al. (2001), Acta Cryst D57:1881-1884. PMID: 11717504 *www.library.arizona.edu/search/subjects/chemistry/biochemjour.html2 Wilson & Brunger (2000), J. Mol. Biol. 301:1237-1256. • 1.0 angstrom resolution: discrete and dynamic disorder in central helix. • Proposal: Ca binding changes dynamics. • What holds it in place? • PDB 1EXR Fallon & Quiocho (2003), Structure 11:1303-1307. • 1.7 angstrom structure of bovine brain calmodulin. • Change in conformation: compact3 Meador, Means & Quiocho (1992), Science 257:1251-1255. 2.4 angstrom complex with peptide from myosin light chain kinase (20 aa, 1 nM binding constant) PDB: PDB 1CDL Horvath et al. (2005), J. Biol. Chem. 280:8266-8274. • Both x-ray and NMR • Complex with anti-tubulin drug KAR-2 • 2.1 angstrom structure • Dynamics? Conformation4 Horvath et al. (2005), J. Biol. Chem. 280:8266-8274. Drum et al. (2002), Nature 415:396-402. • Large complex • Structure Correct? • Conformation of calmodulin? • Contact with Oedema factor? Crystallographic and refinement statistics A. Crystallographic data Crystal EF-CH6-CaM-3'dATP* Space group I222 Cell 117.60, 167.44, 343.48 X-Ray source APS, 14-BM-C Resolution (Å) 20-2.75 completeness (%) 91.6 (57.2) R-sym (last shell) 9.7 (26.4) I/σ (last shell) 4.6 (2.8) Redundancy 10.9 (3.3) R-factor/free (%) 21.5/28.6 Bond length (Å) 0.005 Bond angle (deg.) 1.85 X-ray Summary Oedema Factor – Calmodulin • Drum et al. (2002), Nature 415:396-402. • Drum et al. (2001), Acta Cryst. D57:1881-1884 • What was crystallized? • How were the structures determined? Where’s the good stuff? • Not: Drum et al. (2002) Nature 415:396-402. Nice paper, sparse data. • Yes!: Drum et al. (2001) Acta Cryst. D57:1881-1884. The details of a difficult structure solution.6 What was crystallized? • Recombinant adenylyl cyclase domain of Oedema factor from B. anthracis (EF, residues 290-800), with C-terminal His-tag. Multicolumn purification. • Recombinant Human calmodulin with His-tag. Crystal Forms? • EF-Calmodulin from hanging drops • Just EF (this described in the Nature paper)7 Initial Phases? Used direct methods Phase Improvement – Self-rotation Function (Patterson)8 Final Map EF Structure by Molecular Replacement9 Crystallographic and refinement statistics A. Crystallographic data Crystal EF-CH6-CaM-3'dATP* H6-EF EF-CH6-CaM Space group I222 P21212 I222 a (Å) 117.60 50.47 116.73 b (Å) 167.44 203.60 167.31 c (Å) 343.48 74.03 344.30 X-Ray source APS, 14-BM-C APS, 14-BM-C NSLS X-25 Resolution (Å) 2.75 2.6 2.95 completeness (%) (last shell) 91.6 (57.2) 98.5 (95.4) 98.8 (98.1) R-sym (last shell) 9.7 (26.4) 8.7 (27.3) 6.1 (26.6) I/σ (last shell) 4.6 (2.8) 5.6 (1.7) 18.5 (4.1) Redundancy (last shell) 10.9 (3.3) 6.5 (4.8) 16.0 (5.5) B. Refinement Resolution (Å) 20-2.75 30-2.6 30-2.95 R-factor/free (%) 21.5/28.6 22.8/27.6 27.8/31.5 Bond length (Å) 0.005 0.011 0.011 Bond angle (degree) 1.8 1.55 1.6 Final Statistics – supplement Drum et al. (2002), Nature 415:396-402. Comparison of Calmodulin
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