Monomer structure of nsp4C possess a new fold.

<p>A. Stereo view of the nsp4C monomer Cα backbone trace. Positions of selected residues and the N- and C-termini are labeled. B. Topology of the nsp4C monomer. β-strands are shown in arrows, and α-helices in cylinders. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone-0006217-g002" target="_blank">Fig. 2A</a> was drawn with the programs with PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone.0006217-DeLano1" target="_blank">[47]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone-0006217-g002" target="_blank">2B</a> with TopDraw.</p

Data collection and refinement statistics.

a<p><i>R_merge</i> = Σ_hΣ_l | I_ih−h> |/Σ_hΣ_I h>, where h> is the mean of the observations I_ih of reflection h.</p>b<p><i>R_work</i> = Σ( ||F_p(obs)|−|F_p(calc)||)/Σ|F_p(obs)|; <i>R_free</i>  = R factor for a selected subset (5%) of the reflections that was not included in prior refinement calculations.</p>c<p>Numbers in parentheses are corresponding values for the highest resolution shell.</p

The molecular surface model of the monomer from nsp4C (T408-Q496) WT dimer and mutant C425S.

<p>Electrostatic potential is mapped on the surface, with positive charged region colored in blue and negative charged region in red. Both molecules in one asymmetric unit of the WT nsp4C dimer and mutant C425S are shown in three orientations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone-0006217-g003" target="_blank">Fig. 3A and 3B</a> represents molecule A and molecule B of the WT nsp4C dimer respectively; while <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone-0006217-g003" target="_blank">Fig. 3C</a> represents the monomer of the C425S mutant. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone-0006217-g003" target="_blank">Fig. 3D</a> represents the superposition of molecule A (gold) and B (magenta) from WT and the monomer of the C425S mutant (green), Cys425 is shown in stick representation, with the carbon, nitrogen, oxygen, and sulfur atoms colored yellow, blue, red, and green, respectively; and also the crystal packing of WT nsp4C (magenta), symmetry-related molecules are colored in green, and the molecule forming the equivalent of the C425S mutant dimer is colored in blue. Selected amino acids are labeled, and the figure is drawn by PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone.0006217-DeLano1" target="_blank">[47]</a>.</p

A. Gel filtration analyses of wild-type nsp4C (T408-Q496) and the C425S mutant.

<p>WT, WT with 1mM DTT and C425S were separately loaded in the same quantity and eluted at 0.5 mL min⁻¹ with a buffer containing 50 mM Tris-HCl (pH 8.5), 150 mM NaCl. WT measurements are represented in solid blue line, C425S in medium dashed dark yellow line, and WT containing 1mM DTT in dotted red line. WT was eluted in two peaks corresponding to dimer and monomer respectively; in contrast, C425S and WT containing 1 mM DTT were dominated only by the monomer. B Multiple sequence alignment of MHV-A59 nsp4C (T408-Q496) with the representatives from all three groups of the genus Coronavirus. Key: MHV-A59, Mouse Hepatitis Virus strain A59, NP_001012459; SARS-CoV, Severe Acute Respiratory Syndrome, NP_904322; HCoV-HKU1, Human Coronavirus HKU1, YP_459935; HCoV-OC43, Human Coronavirus strain OC43, NP_937947; BCoV, Bovine Coronavirus, NP_742170; HEV, Porcine Hemagglutinating Encephalomyelitis Virus, YP_459949; HCoV-229E, Human Coronavirus strain 229E, NP_835358; HCoV-NL63, Human Coronavirus strain NL63, YP_003766; PEDV, Porcine Epidemic Diarrhea Virus, NP_598309; TGEV, Transmissible Gastroenteritis Virus, NP_058422; and IBV, Avian Infectious Bronchiolitis Virus, NP_066134. Secondary structural elements of the crystal structure are shown at the top of the alignment; arrows indicate β-strands, and helical curves denote α(or 310)-helices. Residues highlighted in red are identical among the compared proteins; residues highlighted in yellow are conserved. The alignment was generated using the program ClustalX <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone.0006217-Thompson1" target="_blank">[48]</a> and drawn with ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006217#pone.0006217-Gouet1" target="_blank">[49]</a>.</p