N-PilO2_Bp protein.

<p><b>A.</b> Overall fold of N-PilO2_Bp composed of two α/β topology subdomains, each displaying a mixed β-sheet, separated by a (central) cleft. The bound phosphate ion is shown as spheres. <b>B.</b> Topology diagram of N-PilO2_Bp. This diagram was generated using PDBSum server (<a href="http://www.ebi.ac.uk/pdbsum/" target="_blank">www.ebi.ac.uk/pdbsum/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094981#pone.0094981-Laskowski2" target="_blank">[52]</a>. <b>C</b>. Crystal packing of the phosphate-containing N-PilO2_Bp structure, showing the three crystal packing dimers formed by alternative interactions between four symmetry-related monomers (green, blue, magenta and black). The three interfaces are highlighted by black, blue and red shading. The first ‘dimer’, is formed by the interaction between the green (or blue) and the magenta (or black) monomers and the light green (or light blue) phosphate. The second crystallographic dimer occurs between the magenta and black monomers. The third dimer is formed by the green and blue monomers. <b>D</b>. Stereo view of the electron density map for the residues building the phosphate ion binding pocket. The phosphate ion is shown as sphere; the electron density is contoured at 1.5 sigma level. <b>E.</b> Front and back view of N-PilO2_Bp electrostatic surface potential. The electrostatic potential was calculated using the CCP4MG viewer. Negative (red) and positive (blue) charges, and uncharged (white) surfaces are shown. <b>F.</b> Superposition of the 3D structures of N-PilO2_Bp (cyan; PDB codes 4BYZ and 4BZ0) and N-BfpC (chocolate; PDB code 3VHJ).</p

Crystallographic data-collection statistics.

a<p>Data completeness treats Bijvoët mates independently.</p>b<p>Statistics for the highest resolution shells are given in parentheses.</p>c<p><i>R</i>_merge = ∑<i>_hkl</i>∑<i>_i</i>|<i>I(hkl)_I</i> − < <i>I(hkl)</i> >|/∑<i>_hkl</i>∑<i>_i</i>< <i>I(hkl)_i</i> >.</p>d<p>Substructure determination parameters are from ShelxD.</p>e<p>CC  =  [∑<i>wE_oE</i>_c∑<i>w</i> - ∑<i>wE_o</i>∑<i>wE_c</i>]/{[∑<i>w</i>E_o²∑<i>w -</i> (∑<i>w</i>E_o)²] [∑<i>w</i>E_c²∑<i>w</i> -(∑<i>w</i>E_c)²]}^1/2,</p><p>where <i>w</i> is weight. CC_all/CC_weak is the correlation coefficient for all and weak reflections of the best solution.</p>f<p>FOM, figure of merit  =  | <i>F</i>(<i>hkl</i>)best|/|<i>F</i>(<i>hkl</i>)|; <b>F</b>(<i>hkl</i>)best  =  ∑<i>P</i>(α)<b>F</b>_hkl(α)/∑<i>P</i>(α).</p

Comparison of Tfpb machinery R64 thin pilus variant encoding operons for different microorganisms.

<p>The alignment was performed using tblastx from the Blast suite, and visualized in Artemis Comparison Tool. Conserved protein regions are paired by color-shaded regions; the blue and red colors represent the reverse and forward matches, respectively, and color intensity is proportional to the sequence homology. Genes are represented by arrows; the same arrow color indicates putative orthologs. The grey arrows represent genes lacking homologs among represented <i>pil</i> clusters. The <i>pil</i> cluster sequences were retrieved from GenBank: <i>Tfp7</i> locus from <i>B. pseudomallei</i> (<i>Bp</i>) chromosome 2 complete sequence, BX571966.1; PAPI-1 <i>pil</i> gene cluster from <i>P. aeruginosa</i> (<i>Pa</i>) PA14, AY273869.1; R64 transfer region, AB027308.1; and <i>pil</i> operon from <i>Salmonella enterica</i> (<i>Se</i>) subsp. <i>enterica</i> serovar Paratyphi C strain CN13/87, AY249242.1.</p

Composition of the <i>Toxoplasma</i> MARS complex.

<p>Silver-stained PAGE of MARS complex proteins (identified by MS-MS) isolated by FLAG immuno-precipitation of endogenously tagged subunits (indicated in boldface: mF = Myc-FLAG and HF = HA-FLAG epitope tags) from extracellular parasites.</p

Appearance of the MARS complex as revealed by electron microscopy.

<p>(A) Electron micrograph of negatively-stained YRS-HA-FLAG MARS complexes. The scale bar represents 50 nm. (B) Subset of representative propeller views (18/137) of negatively-stained immunoprecipitated YRS-HA-FLAG MARS complexes windowed from images such as shown in (A). (C) Left – Rotational average of all views (1030 particle including the 137 propeller views) following pre-centring. Right – Corresponding radial profile from which the central core (120 Å) and peripheral domain's (240 Å) maximum average diameters can be determined. (D) An average image of aligned representative particles (137 propeller views from B) based on the entire image is shown on the left. On the right, three different class averages (homogenous sub-classes) generated by re-aligning only the central regions of the 137 views on the left are presented. (E) Reference-free class averages (homogenous sub-classes) derived from classification of all windowed particles (1030 in total) not just the propeller views shown in (B). The last image is the rotational average of all particles that went into classification.</p

Localization of the Tg-MARS complex.

<p>(A) Fluorescent, light, and overlay of the two channels of endogenously tagged subunits of Tg-MARS visualised by <i>in situ</i> immunofluorescence in intracellular parasites with anti-Myc or anti-HA (red) antibodies and Hoechst DNA-specific dye (blue). (B) Fluorescent, light, and overlay of two channels of the QRS subunit of Tg-MARS visualised by <i>in situ</i> immunofluorescence in intracellular parasites with an anti-rQRS (red) antibody and Hoechst DNA-specific dye (blue). Scale bar  = 10 µm.</p

Relationship of Tg-p43 to similar amino-acyl tRNA synthetase-interacting multifunctional proteins from other species.

<p>Multiple sequence alignments of the GST-C (GST) and RNA-binding (RB) domains of Tg-p43 and select homologs (Tg = <i>Toxoplasma gondii</i>, Sc = <i>Saccharomyces cerevisiae, Hs = Homo sapiens</i>) are shown above and below a schematic diagram of the Tg-p43 domain arrangement. The residue numbers of domain boundaries are listed in the schematic and at the beginning and end of the individual sequences.</p

Dependence of MARS complex assembly on the scaffold protein Tg-p43.

<p>(A) Silver-stained PAGE gel of immunoprecipitations of HA-FLAG-tagged (HF) YRS and MRS subunits in Tg-p43 knockout strains. (B) Isolation of the MARS complex by immunoprecipitation of a C-terminal-truncated (ΔC) form of the Tg-p43 protein (a.a. 1–296). (C) Western blots of eluted fractions from SEC analyses of crude cell lysates of Tg-p43-containing and knock-out strains (all RHΔ<i>ku80</i>). Corresponding fractions (numbered above the first gel image) are vertically aligned and their solution molecular weights, as determined by calibration with high molecular weight standards, are also given. Molecular weight standards are shown for the anti-FLAG Western only as a reference for evaluation of the knock-out blot.</p

Compositional and size heterogeneity of MARS complex populations.

<p>Silver-stained PAGE gel of eluted fractions of immunoprecipitated MARS complexes separated by SEC. Each panel represents a separate SEC analysis of MARS complexes purified from strains harbouring C-terminal tags on different subunits of the complex: (A) Myc-FLAG-tagged Tg-p43 (mF = Myc-FLAG), (B) HA-FLAG-tagged C-terminal-truncated (ΔC) form of the Tg-p43 (residues 1–296) (HF = HA-FLAG), (C) HA-FLAG-tagged methionyl-tRNA synthetase, and (D) HA-FLAG-tagged tyrosyl-tRNA synthetase. Corresponding fractions (numbered below each gel image, and calibrated by the elution peak of the FLAG peptide) are vertically aligned and their solution molecular weights, as determined by calibration with high molecular weight standards, are given. Grey-levels of the images were adjusted to enhance the contrast but no bands were masked by this process.</p

Structure based sequence alignment of D7 with V_HH A12 and C8 as well as with the V_H domains from the neutralizing antibodies b12, b13, F105 and m18.

<p>The residue numbering is according to Chothia <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010482#pone.0010482-Chothia1" target="_blank">[38]</a> and the CDRs are indicated by coloured bars.</p