Variations in the crystal packing of the MNV NS6^pro A and B chains in the asymmetric unit.

<p>(A) Crystal packing of A and B chains of MNV NS6^pro. The A and B chains of one asymmetric unit are shown along with the neighbouring molecules (labelled A' and B') into which they insert their C-termini. (B) This panel shows the same molecules that are depicted in panel A (with the same colouring) but in this case the A and B chains within the asymmetric unit are superposed; this reveals the very different contacts that they make with their closest neighbour in the crystal. (C) Here the A' and B' chains from panel A are now superposed in order to show the similarity of the conformations of the bound C-termini (shown as sticks) from the A and B chains respectively. Colour-coding is the same as panel A.</p

Cloning and C139A mutagenic primers used in the course of the study.

<p>Restriction sites used in cloning are underlined. Mutations introduced using QuikChange mutagenesis are in boldface.</p

Structural comparison of the MNV NS6 protease with human norovirus NS6^pro and foot-and-mouth disease virus 3C^pro.

<p>(A) Cartoon representation of the MNV NS6^pro structure. The N and C-terminal domains are coloured green and orange respectively. The side-chains of the amino acids that make up the catalytic triad, A139 (mutated from Cys), H30 and D54, are shown as sticks. A disordered loop formed by residues 124–130 (residues 124–131 in chain B) is indicated as a dashed line. The peptide bound to NS6^pro is not shown in this representation. (B) Overlay of HuNV NS6 protease structures from Chiba (PDB-ID: 1WQS), Norwalk (PDB-ID: 2FYQ) and Southampton (PDB-ID: 2IPH) viruses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Nakamura1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Zeitler1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Hussey1" target="_blank">[23]</a>. Excluding the variable C-termini, the root mean square deviations of the backbone atoms of Chiba, Norwalk and Southampton virus NS6^pro from MNV NS6^pro are 0.62, 0.43 and 0.41 respectively. The disordered C-terminus of the Chibavirus protease is shown as a dashed line. (C) Structure of FMDV 3C^pro (PDB-ID: 2J92) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Sweeney1" target="_blank">[26]</a>, coloured as in panel (A).</p

Crystallographic data collection and model refinement statistics for MNV NS6^pro.

1<p>Values for highest resolution shell given in parentheses.</p>2<p>R_merge  = 100 ×Σ_hkl|I_j(hkl) − j(hkl)>|/Σ_hklΣ_jI(hkl), where I_j(hkl) and j(hkl)> are the intensity of measurement j and the mean intensity for the reflection with indices hkl, respectively.</p>3<p>R_work  = 100 ×Σ_hkl||F_obs| − |F_calc||/Σ_hkl|F_obs|.</p>4<p>R_free is the R_model calculated using a randomly selected 5% sample of reflection data that were omitted from the refinement.</p>5<p>RMS, root-mean-square; deviations are from the ideal geometry defined by the Engh and Huber parameters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Engh1" target="_blank">[45]</a>.</p

Sequence conservation of polyprotein junction in MNV that are cleaved by NS6^pro.

<p>(A) The five cleavage junctions of MNV CW1 polyprotein (NCBI accession number YP_720001) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Sosnovtsev1" target="_blank">[14]</a>. (B) Weblogo of polyprotein cleavage junctions cleaved by MNV NS6^pro. This Weblogo was generated using 39 MNV polyprotein sequences and the Weblogo sequence logo generator <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Crooks1" target="_blank">[44]</a>. The height of the letter in each case is indicative of the degree of conservation. The Genbank accession numbers of the other sequences used to prepare the alignment are ABU55618, ABU55627, ABU55615, ABU55621, ABU55612, ABU55624, AEE10026, ABU55600, AEY83582, AEE10023, ABU55606, AEE10020, ABU55609, AEE10017, ABB02416, AEE10002, ACJ72215, AEE09999, ABU55591, AEE10005, ABU55570, AEE10008, ABU55585, AEE10014, ABU55579, AEE10011, ABU55576, ABU55597, ABU55573, ABU55603, ABU55582, ABU55594, ABU55588, ABU55567, ACS70958, ACJ72218, ABS29272, ABS29274.</p

Comparative analysis of protease-peptide interactions for the P6–P1 residues in MNV and SV NS6^pro and CAV 3C^pro.

<p>The N-terminal and C-terminal β-barrel domains of each protease are coloured green and orange respectively. (A) Binding of residues P5–P1 (C-terminus of NS6^pro), shown as sticks colour-coded by atom type (Carbon – light-blue; Oxygen – red; Nitrogen – blue), within the peptide binding grove of MNV NS6^pro. Selected side-chains from the protease are also shown as sticks. Hydrogen bonds and salt-bridges mentioned in the text are indicated by black dashed lines; all such bonds shown are ≤3.1 Å. (B) Same view as in A but showing the surface of MNV NS6^pro. (C) Binding of residues P5–P1 from a peptide-like inhibitor to SV (a human norovirus) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Hussey1" target="_blank">[23]</a>. Water molecules involved in the protease-peptide interaction are shown as red spheres. (D) Same view as in B but showing the surface of SV NS6^pro. (E) The refined σ-weighted 2F_o-F_c map electron density (where F_o and F_c are the observed and calculated structure factors respectively) for an A-chain C-terminal peptide, shown at 1.5 σ. (F) The interaction between residues P6–P1 of a peptide ‘product’ and CAV 3C^pro<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038723#pone.0038723-Lu1" target="_blank">[30]</a>.</p

Acaena observational reproductive output

area, plant identit, shoot identity, number of reproductive shoots per area, individual mean fruit mass, number of fruits per shoot, and total fruit mass per shoot of the cushion plant Acaena integerrima in presence and absence of the species Embothrium coccineu