Immuno-purification of FtsH2-HA.

<p>Intact chloroplasts were first isolated from FtsH2-HA plants, then lysed and solubilized with 1% β-DM. Anti-HA agarose conjugate was added and washed extensively, before elution with 2× sample buffer. Samples from the different steps were resolved by SDS-PAGE, blotted and reacted with HA and FtsH antibodies.</p

Characterization of transgenic plants expressing epitope-tagged FtsH2.

<p>(<b>A</b>) Schematic representation of the HA-tagged FtsH2 construct. A 3×HA tag sequence was cloned in-frame to the FtsH2 cDNA 3′ end. The construct was cloned between the 35S constitutive promoter and the OCS terminator. Arrowheads correspond to primers used in further analyses. (<b>B</b>) PCR on genomic DNA of kanamycin-resistant transgenic plants. Primers A and B (shown in <i>A</i>) were used to distinguish between WT and transgenic plants. (<b>C</b>) Qualitative RT-PCR on total RNA. Primers A and B were used to detect FtsH2-HA transcripts. Primers A and C were used to detect the native FtsH2 transcript. All other transgenic seedlings demonstrated a similar behavior. (<b>D</b>) Immuno-blot analysis of total protein from FtsH2-HA and WT plants. The blot was reacted with an HA antibody. Load is 10 µg chlorophyll per lane. All other transgenic seedlings demonstrated a similar behavior.</p

Relative abundance of FtsH subunits.

<p>The abundance of FtsH subunits was deduced from the peak area of specific peptides identified in the MS analysis. (<b>A</b>) Ratios between ‘type B’ and ‘type A’ subunits in isolated complexes (C) and intact thylakoids (T). The Roman numerals correspond to those indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036008#pone-0036008-g004" target="_blank">Figure 4</a>. The horizontal dashed lines represent possible ratios within the hexamer. (<b>B</b>) Ratios between the products of duplicated genes within a type. The sequences of the peptides used in the analysis are indicated above the bars. Values are averages ±s.d. of 2–4 replicates.</p

Abundance and localization of the FtsH2-HA protein.

<p>(<b>A</b>) Immuno-blot analysis of total protein, extracted from transgenic and WT plants. Antibodies used are indicated on the left. (<b>B</b>) Intact chloroplasts were isolated from FtsH2-HA plants and fractionated into thylakoids and stroma. Samples were separated by SDS-PAGE, and gels were either stained by Coomassie blue or blotted onto membranes and reacted with anti-HA and anti-FtsH antibodies. All samples contained equivalents of 3 µg chlorophyll, except the HA blot, which contained equivalents of 10 µg chlorophyll. The antibodies used, or the location of specific proteins on the stained gels, are indicated on the left.</p

Mass spectrometry analysis of purified FtsH2-HA.

<p>Alignment of mature FtsH1, FtsH5, FtsH2 and FtsH8. Highlighted are peptides identified by MS. Yellow, peptides conserved between FtsH1 and FtsH5; green, peptides conserved between FtsH2 and FtsH8; blue, peptides specific to FtsH1; purple, peptides specific to FtsH5; olive, peptides specific to FtsH2; gray, peptides specific to FtsH8. Red Roman numerals indicate peptide groups used for quantification.</p

Models of the <i>Arabidopsis thaliana</i> FtsH complex.

<p>(<b>A</b>) TtFtsH (left), structure of <i>T. thermophilus</i> (PDB entry 2DHR) complex rendered as solvent accessible surface. Shown is the face of the protease side, with each chain in a different color. Models of the AtFtsH2 (middle) and AtFtsH2/5 rendered as TtFtsH. Chains of AtFtsH2 are colored in blue and chains of AtFtsH5 are colored in orange. (<b>B</b>) Summary of the calculated buried surface interfaces of the monomers in the <i>T. thermophilus</i>, AtFtsH2 and AtFtsH2/5 complexes. To facilitate the comparison, the values are color-coded as in <i>A</i>. (<b>C</b>) Zoom-in at the interface between chains <i>b</i> and <i>c</i>. The left and the right panels depict the homomeric (FtsH2) and the heteromeric (FtsH2/FtsH5) complexes, respectively. The picture in the left panel is centered at Arg-204 in chain <i>b</i> (marine blue), and its nearby residues in chain <i>c</i> (dark blue). Chain <i>c</i> of FtsH5 is colored in orange (like in 6A). Yellow dashed lines denote salt bridges; distances are indicated in Angstroms. Color code: oxygen – red; nitrogen – blue; carbon – yellow.</p

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p

Absence of a long-term trend in <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).

<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p

Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.

<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p