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. 2012 Apr;68(Pt 4):391-403.
doi: 10.1107/S090744491104978X. Epub 2012 Mar 16.

Application of DEN Refinement and Automated Model Building to a Difficult Case of Molecular-Replacement Phasing: The Structure of a Putative Succinyl-Diaminopimelate Desuccinylase From Corynebacterium Glutamicum

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Free PMC article

Application of DEN Refinement and Automated Model Building to a Difficult Case of Molecular-Replacement Phasing: The Structure of a Putative Succinyl-Diaminopimelate Desuccinylase From Corynebacterium Glutamicum

Axel T Brunger et al. Acta Crystallogr D Biol Crystallogr. .
Free PMC article

Abstract

Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.

Figures

Figure 1
Figure 1
Primary-sequence alignment between 1vgy (chain A) and Cgl1109. The alignment obtained by PROMALS3D (Pei et al., 2008 ▶) is shown. The first line in each block shows conservation indices for positions with a conservation index above 4. The last two lines show consensus amino-acid sequence (Consensus_aa) and consensus predicted secondary structure (Consensus_ss). The representative sequences are named in magenta and are colored according to predicted secondary structure (red, α-helix; blue, β-strand). The first and last residue numbers of each sequence in each alignment block are shown before and after the sequences, respectively. Consensus-predicted secondary-structure symbols: α-helix, h; β-strand, e. Consensus amino-acid symbols are as follows (conserved amino acids are shown in bold uppercase letters); aliphatic (I, V, L), l; aromatic (Y, H, W, F), @; hydrophobic (W, F, Y, M, L, I, V, A, C, T, H), h; alcohol (S, T), o; polar residues (D, E, H, K, N, Q, R, S, T), p; tiny (A, G, C, S), t; small (A, G, C, S, V, N, D, T, P), s; bulky residues (E, F, I, K, L, M, Q, R, W, Y), b; positively charged (K, R, H), +; negatively charged (D, E), −; charged (D, E, K, R, H), c. Note that the sequence numbers refer to the genomic sequence of Cgl1109 (taking into account the minor mutations in the construct used for crystallization; see text) and 1vgy. The residue numbering in the deposited PDB file (PDB entry 3tx8) begins with the first residue of the expression construct used, so it is offset by 11 residues compared with the genomic sequence.
Figure 2
Figure 2
Interaction between symmetry-related molecules. A primary molecule (orange) and the nearest symmetry-related molecules (blue) obtained by applying the symmetry operators of the space group of the crystal (P6522) to the primary molecule are shown, as well as lattice translations. Taken together, all these molecules form a network of interactions which is connected throughout the crystal in all three dimensions. The molecules interact through three interfaces, labelled 1, 2 and 3. Interface 2′ is related by crystallographic symmetry to interface 2. Of the three interfaces, interface 1 involves the most extensive interactions, with a buried suface area of 1569 Å2 (compared with 541 Å2 for interface 2 and 276 Å2 for interface 3; the buried surface areas were computed with the PDBePISA server). Considering the extensive interactions, interface 1 is likely to promote dimerization of the molecule, as is also suggested by the PDBePISA server.
Figure 3
Figure 3
DEN refinement starting from molecular-replacement solution. The best R free value for each parameter pair (γ, w DEN) among 20 repeats is shown; for each parameter pair we performed 20 repeats of the DEN-refinement protocol consisting of ten macrocycles of torsion-angle refinement and restrained individual B-factor refinement (for details, see text). The R free value is contoured using values calculated on a 6 × 6 grid (marked by small + signs) where the parameter γ is (0.0, 0.2, 0.4, 0.6, 0.8, 1.0) and w DEN is (0, 3, 10, 30, 100, 300); the results for w DEN = 0 (i.e. torsion-angle refinement without DEN restraints) are independent of γ and the same value was used for all grid points with w DEN = 0. The value of R free varies from 0.444 to 0.479. The contour plot shows two pronounced minima in the range 300 ≥ w DEN ≥ 100, with the absolute minimum at w DEN = 300, γ = 0.2.
Figure 4
Figure 4
Comparison of various refinements and maps for residues 66–77. The sequence numbers refer to the genomic sequence of Cgl1109 (see Fig. 1 ▶). (a) Standard refinement (gray) versus the final model (orange). (b) Standard refinement and one round of AutoBuild (blue) versus the final model (orange). (c) DEN refinement (green) versus the final model (orange sticks). (d) DEN refinement and one round of AutoBuild (magenta) versus the result of semi-automated rebuilding (yellow) versus the the final model (orange). (e) 2mF oDF c electron-density map after standard refinement (blue mesh) and a subsequent round of AutoBuild (cyan mesh) versus the final structure (orange sticks). (f) 2mF oDF c electron-density map after DEN refinement (blue) and a subsequent round of AutoBuild (cyan) versus the final structure (orange sticks). (g) Electron-density map obtained by density modification of the MAD map (blue) versus the final structure (orange sticks). (h) 2mF o − DF c electron-density map (blue mesh) of the final model (orange sticks).
Figure 5
Figure 5
Comparison of various refinements and maps for residues 251–276. Residues 251–263 comprising an α-helix, residues 264–271 comprising a loop and residues 272–276 comprising a β-strand are shown (the sequence numbers refer to the genomic sequence; see Fig. 1 ▶). (a) Standard refinement (gray) versus the final model (orange). Standard refinement produces fragmented or incorrectly connected electron density (marked by arrows). (b) Standard refinement and one round of AutoBuild (blue) versus the final model (orange). Electron density is still fragmented or shows incorrect connectivity. (c) DEN refinement (green) versus the final model (orange). (d) DEN refinement and one round of AutoBuild (magenta) versus the result of semi-automated rebuilding (yellow). (e) 2mF oDF c electron-density map after standard refinement (blue mesh) and a subsequent round of AutoBuild (cyan mesh) versus the final structure (orange sticks). (f) 2mF o − DF c electron-density map after DEN refinement (blue) and a subsequent round of AutoBuild (cyan) versus the final structure (orange sticks). (g) Electron-density map obtained by density modification of the MAD map (blue) versus the final structure (orange sticks). (h) 2mF o − DF c electron-density map (blue mesh) of the final model (orange sticks).
Figure 6
Figure 6
Comparison of various refinements and maps for residues 251–276. A close-up view of the loop consisting of residues 264–271, which is also part of Fig. 5 ▶, is shown. The final model is colored orange (sticks and cartoon representation). The structure after the first round of DEN refinement and AutoBuild is colored magenta (sticks and cartoon representation) and the corresponding 2mF oDF c electron-density map (with model phases calculated from this structure, but without experimental phase information, and contoured at 1.4σ) is colored marine blue. The electron-density map clearly shows that the loop needed to be corrected.
Figure 7
Figure 7
Significance of selenium sites. The standard deviation above the mean (σ) in anomalous difference Fourier maps is shown for the six selenium sites of the SeMet variant of Cgl1109. For comparison, the standard deviation of the highest noise peak is also shown. The amplitudes for the calculation of the anomalous difference Fourier map were obtained from the diffraction data at the peak wavelength (Table 1 ▶). The phases were obtained from the atomic model after standard refinement (blue diamonds), standard refinement followed by automated building with AutoBuild (green triangles), DEN refinement (yellow squares) and DEN refinement followed by automated model building with AutoBuild (red circles).
Figure 8
Figure 8
Comparison of Cgl1109 with 1vgy-A. A superposition of the final model of Cgl1109 (orange cartoon) and chain A of PDB entry 1vgy (blue cartoon) is shown. The superposition was performed with PyMOL (DeLano, 2002 ▶).

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