Experimental workflow and chemical structure and properties of the crosslinking agents employed.

<p><b>A,</b> schematic representation of the experimental workflow used for growing cells, preparing microsomes, carbonate wash of membranes, chemical crosslinking of proteins and preparation of peptides for MS analysis. <b>B,</b> chemical structure and reactivity of the crosslinkers employed BS3 (bis[sulfosuccinimidyl] suberate) and EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.</p

Molecular mass and scores of XLs found by pLink for Pma1p and BSA.

<p>Distributions of scores and calculated molecular masses of XLs generated by pLink by scanning through all mgf files from BS3 experiments (<b>A-D</b>) or EDC experiments (<b>E-H</b>) for Pma1p and BSA, a protein that is not present in the sample, are plotted as calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s007" target="_blank">S7 Table</a>. The cut off lines used to filter all pLink XLs (stippled red lines) indicate a minimal molecular mass of XL of 1’500 Da and scores of ≤ 7.5 x 10⁻⁴ for BS3-XLs, or ≤ 4 x 10⁻⁴ for EDC-XLs.</p

Frequency of XLs found by pLink for a given protein is correlated with its copy number.

<p>The number of XLs for 18 arbitrary chosen MSPs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s004" target="_blank">S4 Table</a> is plotted as a function of the copy number of the respective MSP (<b>A</b>) or its amino acid length (<b>B</b>). In panel <b>C</b>, the number of BS3- and EDC-generated XLs for these 18 proteins are plotted separately, as a function of copy number. In panel <b>D</b>, the distance in the primary sequence between crosslinked amino acids in 160 XLs found in 27 proteins of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s014" target="_blank">S14 Table</a> are plotted.</p

BS3 and EDC crosslinks mapped onto the structural models of Pma1p, Por1p and Sec61p.

<p>Structures of Pma1p (<b>A, B</b>), Por1p (<b>C-F</b>), and Sec61p (<b>G, H</b>) were homology modeled by HHPRED [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref015" target="_blank">15</a>] using plasma membrane H⁺-ATPase from <i>Neurospora crassa</i> (1mhs_A) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref016" target="_blank">16</a>], the voltage-dependent anion channel VDAC1 from <i>Mus musculus</i> (4c69_X) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref017" target="_blank">17</a>], and Sec61 from <i>Canis lupus/Bos Taurus</i> (3jc2_1) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref018" target="_blank">18</a>] as template. Structural models were visualized by PyMOL and the position of crosslinks connecting the Cα atoms of amino acids were added manually based on the experimental data from pLink.</p

BS3 crosslinks mapped onto the topological model of Fks1p, the catalytic subunit of 1,3-beta-D-glucan synthase.

<p><b>A,</b> Arch model of Fks1p indicating the positions of BS3 (red) XLs visualized using xVis. <b>B,</b> topology model of Fks1p proposed using the crosslinking data to validate a topology prediction visualized by Protter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref019" target="_blank">19</a>].</p