Kammerer et al. 10.1073/pnas.0502390102. |
Fig. 6. Biophysicaland biochemical analysis of mutant coiled-coil proteins. (A) Sedimentation equilibrium analysis of 0.76 mg/ml ccCor1 (black), ccCor1-R450A (red), and ccCor1-R450Nle (blue) in PBS at 20°C and 26,000 rpm. The lines were obtained by fitting the data with a single ideal species model. (B and C) SDS/PAGE analysis of recombinant TN-C-p1C64S/R125K (B) and ccMat1-R487A (C) mutant proteins under reducing (r) and nonreducing (n) conditions. Positions of molecular mass markers (in kDa) and disulfide-linked species are indicated on the left and right, respectively, of each gel.
Fig. 7. Sequence alignment of intracellular (I), extracellular (II), transmembrane (III), viral (IV), and synthetic (V) proteins containing short three-stranded coiled-coil domains. Protein families are: Ia, coronins; Ib, heat shock factor-binding protein; IIa, tenascins; IIb, matrilins; IIc and III, collagens; IVa, lentivirus envelope proteins; IVb; influenza hemagglutinine; IVc, coronavirus glycoproteins; V, synthetic coiled coils ccb-p (1) , p1 and p4 (2, 3), and GCN4p-Cort T/L (4). Heptad repeats are shown in blocks of seven amino acid residues. Residues at heptad positions a and d are underlined. The conserved residues forming the trimerization motif seen in ccCor1 are shown in bold and colored according to their physicochemical properties: blue, positively charged; red, negatively charged; green, hydrophobic. Sequences are identified by names and species (h, human; m, mouse; r, rat; ch, chicken; b, bovine; dd, Dictyostelium discoideum; dr, Danio rerio; xl, Xenopus; ce, Caenorhabditis elegans; sc, Saccharomyces cerevisiae; sp, Schizosaccharomyces pombe; dm, Drosophila melanogaster; at, Arabidopsis thaliana). The deduced consensus sequence is shown at the bottom.
1. Kammerer, R. A., Kostrewa, D., Zurdo, J., Detken, A., Garcia-Echeverria, C., Green, J. D., Muller, S. A., Meier, B. H., Winkler, F. K., Dobson, C. M., et al.. (2004) Proc. Natl. Acad. Sci. USA 101, 4435-4440.
2. Burkhard, P., Meier, M. & Lustig, A. (2000) Protein Sci. 9, 2294-2301.
3. Burkhard, P., Ivaninskii, S. & Lustig, A. (2002) J. Mol. Biol. 318, 901-910.
4. Kammerer, R. A., Schulthess, T., Landwehr, R., Lustig, A., Engel, J., Aebi, U. & Steinmetz, M. O. (1998) Proc. Natl. Acad. Sci. USA 95, 13419-13424.
Table 3. Crystallographic data, refinement statistics, and overall coiled-coil geometry
Space group | SLS P3 data set | In-house P1 data set | SLS P1 data set |
P3 (No. 143) | P1 (No. 1) | P1 (No. 1) | |
Unit cell axes a, b, c | 39.8 Å, 39.8 Å, 46.6 Å | 23.7 Å, 23.7 Å, 46.4 Å | 23.6 Å, 23.6 Å, 46.4 Å |
Resolution range | 47.0 – 1.20 Å | 20.0 – 1.86 Å | 20.0 – 1.20 Å |
No. of observed reflections | 59,566 | 18,468 | 92,239 |
No. of unique reflections | 24,824 | 6,653 | 24,694 |
R sym total / outer shell* | 0.059 / 0.20 (1.25 – 1.20 Å) | 0.023 / 0.033 (1.93 – 1.86 Å) | 0.056 / 0.14 (1.24 – 1.20 Å) |
Completeness total / outer shell | 0.96 / 0.81 (1.25 – 1.20 Å) | 0.91 / 0.80 (1.93 – 1.86 Å) | 0.92 / 0.81 (1.24 – 1.20 Å) |
No. of refined nonhydrogen atoms: protein / water / Zn2+ |
|
|
|
R factor / RFree-R factor† | — | — | 0.16 / 0.20 |
rmsd bond lengths / angles‡ | — | — | 0.015 Å / 1.5° |
*
Rsym = ShSi | Ii(h) – <I(h)> | / ShSi Ii(h) , where Ii(h) and <I(h)> are the ith and mean intensity over all symmetry-equivalent reflections h.†
R = S | FC – FO | / S FO , where FC is the calculated structure factor amplitude of the model, and FO is the observed structure factor amplitude.‡
rmsd, rms deviation from ideal stereochemistry.Superhelical parameters
(1): coiled-coil radius, 6.43 Å; coiled-coil rise,1.45 Å; coiled-coil pitch, 148.5 °.1. Strelkov, S. V. & Burkhard, P. (2002) J. Struct. Biol. 137, 54-64.