Transthyretin Eiger 9M X-ray diffraction dataset

<p>Transthyretin X-ray diffraction dataset collected during commissioning of Eiger 9M detector on Proxima2A beamline, Synchrotron SOLEIL, France.</p

MOESM1 of Structural and functional characterization of a highly stable endo-β-1,4-xylanase from Fusarium oxysporum and its development as an efficient immobilized biocatalyst

Additional file 1. Additional Fig. 1. Schematic representation of Xyl2 topology. Additional Fig. 2. Docking of a xylose hexaoligosaccharide on Xyl2 (pH 5). Additional Fig. 3. Schematic representation of the rationale for random enzyme immobilization via the carrier or carrier-free approaches. Negative correlation between Xyl2 activity yield and functionalization degree in high and low agarose supports. Additional Table 1. Guiding values for binding capacities of commercial agarose beads employed for Xyl2 immobilization

Interaction network for ct⁶A37-tRNA^ANN modification.

<p>TcdA (surface representation, monomer chains in green and wheat colors) interacts transiently but specifically with the sulfur acceptor CsdE (in grey, with TcdA-binding surface patches in blue and Cys61 in yellow), linking with the CsdA-CsdE cysteine desulfurase system and sulfur trafficking, which are known to be required for ct⁶A37 synthesis <i>in vivo</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118606#pone.0118606.ref016" target="_blank">16</a>]. The CsdE-TcdA transient interaction is represented by a grey double-headed arrow. TcdA interacts with tRNA^ANN (<i>K</i>_D in the μM range) in a 2:2 complex that harbors ATP-dependent t⁶A37 dehydratase activity. The ct⁶A37 hypermodification is important for decoding efficiency and translation fidelity.</p

TcdA interacts transiently with the sulfur-acceptor SufE-like protein CsdE.

<p><b>a</b> NMR ¹H-¹⁵N NOESY spectrum of doubly labeled ¹³C,¹⁵N-CsdE in presence (blue) or absence (black) of unlabeled TcdA. Significant shifts in the position of specific NOESY resonance peaks for CsdE residues are labeled. <b>b</b> The position of the affected amino acid residues is mapped onto the NMR structure of CsdE (PDB 1NI7) in ribbon (left) and surface (right) representations. <b>c</b> Average chemical shift (Δδ) of ¹³C,¹⁵N-CsdE upon binding to TcdA as measured from the NOESY spectrum in <b>a</b> plotted against the CsdE amino acid sequence.</p

Structural comparison of TcdA with the homologous E1-like activating enzymes.

<p><b>a</b> Ribbon representation of TcdA structural homologs found with PDBeFold [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118606#pone.0118606.ref051" target="_blank">51</a>] with structural similarity Q-scores higher than 0.28 (the highest Q-scores are 0.49 with MoeB and 0.46 with ThiF). All structures were superimposed onto TcdA and are shown in similar orientations for comparison: MoeB (PDB 1JWB), ThiF (PDB 1ZFN), MccB (PDB 3H9J) and UBA5 (PDB 3H8V). E1-like core domain is shown in grey; the long α-helix that is structurally equivalent to TcdA α8 is in orange; metal binding sites are in protein-specific colors; K⁺ ions are in purple, Na⁺ ions in yellow, and Zn²⁺ in grey. <b>b</b> Schematic representation of the domain architecture of the E1-like enzymes superimposed in (<b>a</b>), with equivalent color coding; bs, binding site; the sequence position corresponding with disordered loops is indicated by dashed lines and marked with start and end residues. The C-terminal α8 helix in TcdA is structurally equivalent to the α-helix immediately following the E1-like domain in the other enzymes. <b>c</b> Structure-guided multiple sequence alignment of the E1-like domain with overlaid secondary structure from TcdA. Functionally important residues and motifs are annotated as follows: bold underline, P-loop residues; black asterisks, residues in direct contact with ATP; and blue asterisks, residues from helix H3₁₀a that make water-mediated contacts with the nucleotide. Conserved residues are shown in shaded colors.</p

TcdA-tRNA interface.

<p><b>a</b> Electrostatic potential molecular surface calculated with APBS (Adaptive Poisson-Boltzmann Solver) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118606#pone.0118606.ref055" target="_blank">55</a>] and rendered with PyMOL (<i><a href="http://www.pymol.org" target="_blank">www.pymol.org</a></i>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118606#pone.0118606.ref056" target="_blank">56</a>]. Two views are shown that are related by a 90° rotation around a horizontal axis. The interfacial Na⁺ cation is depicted as a yellow sphere. <b>b</b> tRNA is modeled on the basis of the SAXS data for the TcdA-tRNA^Lys(UUU) complex bound to the two outer rims of TcdA (represented as in <b>a</b>), where most of the positively charged surface is found. The two tRNA molecules bind to spatially separated and independent surface patches in a symmetric arrangement. The modified ct⁶A37 nucleotide is shown in cyan. <b>c</b> Detailed view of the TcdA-tRNA binding mode. TcdA is represented as in (<b>a</b>), with Cys66 sulfur atom shown as a green sphere. The tRNA molecule on the front inserts its anticodon-stem loop into the ATP-binding pocket (in spheres and CPK colors), with ct⁶A37 (cyan) facing the catalytic site.</p

Crystallographic data processing and refinement statistics.

<p>^aRmeas = Σ_hkl (n/n-1)^1/2 Σ_i |I_i(hkl)-| / ΣΣ_i I_i(hkl); where i is the ith measurement of reflection (hkl) and is the average over symmetry related observations of a unique reflection (hkl).</p><p>^bCC1/2 is the Pearson correlation coefficient calculated between two random half data sets.</p><p>^cCC* is the CC of the full data set against the true intensities, estimated from CC* = [2 CC1/2/(1+CC1/2)]^1/2.</p><p>Crystallographic data processing and refinement statistics.</p

X-ray electron density maps of the ATP- and AMP-binding pocket and metal coordination spheres.

<p><b>a</b> Detailed view of the ATP-binding site of TcdA. Residues that interact with ATP are labeled and shown as sticks and atom colors (C atoms are in subunit colors, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118606#pone.0118606.g001" target="_blank">Fig. 1</a>). 2mF_o-DF_c electron density map is depicted around the ATP substrate at 1.5 σ contour level. <b>b</b> Detailed view of AMP bound in the active site. Representation and electron density map as in (<b>a</b>). <b>c</b>, <b>d</b> Metal coordination spheres of K⁺ (<b>c</b>) and Na+ (<b>d</b>) cations. Interacting residues are labeled and shown as sticks in atom colors (C atoms are in subunit colors), and coordinating water molecules are shown as red spheres. Metals and their coordination spheres are shown in 2mF_o-DF_c electron density (grey). The anomalous Fourier map calculated from a long wavelength (1.89 Å) dataset at 2.4-Å resolution is shown for K⁺ (<b>c</b>) in purple; in contrast, Na⁺ has no anomalous signal at that wavelength, enabling the accurate discrimination between K⁺ and Na⁺ cations in the structure.</p

TcdA dimerization interface.

<p>Molecular surface representation of a TcdA dimer in two orientations (<b>a</b>) related by a 90° rotation around a horizontal axis. The location of the ATP binding pocket and the interfacial Na⁺ ion are labeled. Amino acid residues that contribute to the extended, flat dimer interface are mapped onto the molecular surface in green and labeled. <b>b</b> Close-up of the symmetric helical bundle (H3-H4). Key hydrogen bonding and charge interactions are shown as dashed lines. <b>c</b> Helix H1 participates in the dimer interface through hydrogen bonding and van der Waals interactions with H3 from the same chain and H3₁₀a from the opposite monomer.</p

Solution structure of TcdA-tRNA^Lys(UUU).

<p><b>a</b> SAXS data for TcdA-tRNA^Lys was measured using an online HPLC setup to separate complex from excess free tRNA. The graph shows a plot of the SAXS intensity at zero angle, I(0) (left axis, curve represented as a solid black line), and of the radius of gyration, R_g (on the right axis), versus data-collection frames. Frames 253–263 (green line) were merged and used for shape restoration of the TcdA-tRNA^Lys(UUU), and frames 304–310 were used for the control reconstruction of the tRNA^Lys(UUU) shape. <b>b</b> Best model calculated for the TcdA-tRNA^Lys(UUU) complex overlayed by the ab initio SAXS shape calculated with DAMMIF. The crystal structure of TcdA-ATP is represented in green cartoon and the tRNA is depicted with its main chain as a gold ribbon and the bases as ladders. The fit (red line) to the experimental SAXS data (blue points), calculated with CRYSOL, χ² and residuals are shown. <b>c</b> Like in (<b>b</b>), for free tRNA^Lys(UUU). In this case, the model is a rigid-body fit of the tRNA structure into the ab initio SAXS envelope.</p