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1.
Figure 1.

Figure 1. From: Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain.

Structure of the catalytically inactive tTruB(D48N)–RNA complex. (A) Chemical structure of 5-fluorouridine (f5U). (B) Chemical structure of the enzymatic reaction product, 5,6-dihydro-6-hydroxy-5-fluoro-pseudouridine. Formally, this results from reattachment of the base to the sugar through C5, and addition of water to the C5–C6 double bond (Spedaliere et al. 2004). (C) Omit σA |Fo|−|Fc| electron density in the active site of the tTruB(D48N)–RNA complex (mesh) calculated with phases from a model missing the nucleobase of RNA residue 55 as well as the Cγ, Oδ, and Nδ atoms of residue 48. The map was contoured at 3.5 standard deviations above mean peak height (SD). The nucleobase electron density approximates an oblate ellipsoid whose long axis is parallel to the glycosidic bond, demonstrating that the base remains planar. (D) Omit σA |Fo|−|Fc| electron density around the isomerized RNA residue 55 of the wild-type tTruB complex (PDB code 1K8W). The map was calculated at 2.8 Å resolution, and contoured at 3.5 SD (cf. planar nucleobase in C). (E) Residual |Fo|−|Fc| electron density map (3.5 SD) calculated with a model from which the fluorine atom of residue 55 (in the anti conformation; torsion angle=150°) has been omitted. Once the fluorine atom is added to density feature 1, there are no residual electron density features in the active site except for the noise peak (labeled 2) next to the ribose. (F) Superposition of the two structures. The side chains and nucleotide 55 of the wild-type complex (gray) are labeled in parentheses.

Charmaine Hoang, et al. Protein Sci. 2005 August;14(8):2201-2206.
2.
Figure 2.

Figure 2. From: Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain.

Small active-site perturbations propagate preferentially to the substrate-binding thumb of TruB. (A) Worm representation of tTruB onto which the magnitude of the difference in Cα position between the superimposed wild-type and mutant RNA complexes has been mapped in shades of blue. The darkest blue (at the poorly ordered N terminus) denotes a displacement of ~1 Å. The darkest residues in the thumb have Cα displacements of ~0.7 Å. The active site loop (where the D48N mutation lies) and motif I have displacements of ~0.4 Å and ~0.5 Å, respectively. (B) View orthogonal to A. (C) Ribbon representation of the tTruB–RNA complex. The thumb is in blue, the rest of the protein in gray, the RNA in yellow, with the site of modification (nucleotide 55) in red. The active site residue R181 (A) is located at the N-terminal (upper) end of helix α5. (D) Cα representation of the tTruB–RNA complex (PDB code 1K8W), color-coded by crystallographic thermal parameter. The parameter ranges from 15 Å2 (dark blue) to 68 Å2 (red). (E) Detail of the interaction of the thumb with the active site. The thumb of the wild-type (dark blue) and mutant (light blue) complexes is shown as a ribbon. In yellow is the backbone of residues 126–128. Hydrogen bonds between backbone atoms and the active site arginine (R181), as well as the phosphate 3′ to RNA residue 55 are shown, as is a hydrogen bond between the precursor nucleobase and R181. “HFΨ” refers to the isomerization product (Fig. 1B ▶) of 5-fluorouridine. The RNA-thumb and the R181-thumb interactions highlighted are nearly identical between the E. coli (Hoang and Ferré-D’Amaré 2001) and Thermotoga maritima (Chaudhuri et al. 2004; Phannachet and Huang 2004) TruB-RNA complex structures.

Charmaine Hoang, et al. Protein Sci. 2005 August;14(8):2201-2206.

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