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The alignment used for the calculations of the double-domain sequences tree. (FASTA 53 kb

Additional file 6: of Phylogenomics and sequence-structure-function relationships in the GmrSD family of Type IV restriction enzymes

Results of the positive/negative selection patterns analysis. (DOCX 139 kb

Additional file 5: of Phylogenomics and sequence-structure-function relationships in the GmrSD family of Type IV restriction enzymes

The alignment used for the calculations of the double-domain sequences tree. (FASTA 53 kb

Additional file 3: of Phylogenomics and sequence-structure-function relationships in the GmrSD family of Type IV restriction enzymes

The alignment of GmrS domain sequences used for tree calculations. (FASTA 38 kb

Plots of the fluctuations of the α-carbon atoms as obtained by the UNRES simulation.

<p>The secondary-structure elements are shown above the abscissa. The black, thin line corresponds to the β factors as obtained from the HP0377 (PDB: 4FYC) structure. The simulation results are marked as follows: wild-type HP0377 (black “WT”); CSYC, LcP, (red, “LcP”); CSYC, TcT, (green, “TcT”). E95 and Q95 are colored blue and purple, respectively.</p

Analysis of HP0377-apocytochrome c complex formation.

<p><b>(A) Size exclusion profile of the HP0377, apocytochrome, HP0377-apocytochrome complex and HP0377-apocytochrome complex treated with DTT separated on an Enrich</b>^™<b>sec70 column (Biorad) and monitored by absorbance at 280 nm</b>. HP0377 elutes as a peak at 10.7 min, with an estimated mass of 23.5 kDa, consistent with the size of the monomer. Apocytochrome elutes at 12.9 min, with estimated mass of 9.5 kDa. Complex eluted at 8.9 min, with estimated mass of 56 kDa. Complex treated with DTT elutes as two peaks at 12.9 min and 10.7 min, consistent with the sizes of the HP0377 and apocytochrome. The column was calibrated with Gel Filtration Standard (Bio-Rad): Thyroglobulin (670 kDa), γ-globulin (158 kDa), Ovalbumin (44 kDa), Myoglobin (17 kDa), Vitamin B12 (1.35 kDa). The relative positions of the chosen standards are marked with arrows. <b>(B) SDS-PAGE analysis of HP0377-apocytochrome complex formation</b>. Complexes formed between HP0377_CSYA and apocytochrome were first purified using Ni-NTA resin, and then separated on a size exclusion column. The samples were next analyzed by SDS-PAGE with or without DTT.</p

Cytochrome <i>c</i> oxidation activities of membranes from different <i>B</i>. <i>subtilis</i> strains.

<p>Cytochrome <i>c</i> oxidation activities of membranes from different <i>B</i>. <i>subtilis</i> strains.</p

HP0377 truncated variants act as wild type HP0377.

<p><b>(A)<i>In vitro</i> isomerase activity assay</b>. The reaction contained 40 μM scrambled RNase in 200 mM potassium phosphate buffer, pH 7.0, 2 mM EDTA, 20 μM DTT, and 9 mM cCMP. The reaction was performed in the absence or presence of 20 μM EcDsbC, 20 μM HP0377 and its variants. The cleavage of cCMP by refolded RNase was monitored continuously at 296 nm. The changes in the absorbance at 296 nm as a function of time are presented. Three independent experiments were performed. <b>(B) <i>In vitro</i> reductase assay of HP0377 variants towards apocytochrome c (HP1227)</b>. Two different SDS PAGEs were run to better visualize the shift between oxidized and reduced forms of proteins. Different redox forms were detected by nonreducing SDS-PAGE after AMS treatment, which results in an increase in the molecular mass of reduced proteins by about 0.5 kDa per thiol group. <b>(C) Glutaraldehyde crosslinking of truncated versions of HP0377. 1) N-terminal-shortened HP0377; 2) C-terminal-truncated HP0377</b>. Purified HP0377 truncated versions at 2.5 mg/ml were cross-linked in the presence of different concentration of glutaraldehyde: 1) purified HP0377 protein, 2) 0.001%, 3) 0.005%, 4) 0.01%, 5) 0.05%, 6) 0.1% glutaraldehyde. M—monomers, D—dimers.</p

Phylogenetic tree of thioredoxin domains from HP0377 and related proteins.

<p>Four clades are indicated on the ring surrounding the tree. Numbers correspond to branch support values provided by the FastTree algorithm. Relevant sequences are highlighted and their names are shown in curly brackets.</p

Primers used in this study.

<p>Primers used in this study.</p