D xylose

Xyl crystals were obtained at 283 K using the hanging-drop vapor-diffusion method under conditions similar to those previously described [23 (link)]. In a typical experiment, 1 μl of Xyl solution was mixed with an equal volume of reservoir solution containing 0.1 M HEPES (N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid)) buffer, pH 7.0, and 5% (w/v) PEG 6000. Crystal formation was accelerated by streak seeding using available Xyl crystals as seeds. In these conditions, tetragonal Xyl crystals with dimensions of about 100 x 100 x 100 μm were formed overnight.
Prior to data collection, a Xyl crystal was immersed for 5 s in reservoir solution containing 20% (v/v) glycerol before flash-cooling in liquid nitrogen. Xyl crystals with bound L-arabinose (Xyl•arabinose), D-xylose (Xyl•xylose), or L-arabinose and D-xylose (Xyl•arabinose•xylose) were prepared by soaking Xyl crystals for 10 min in reservoir solution supplemented with 500 mM L-arabinose (Sigma-Aldrich), D-xylose (Sigma-Aldrich), or both L-arabinose and D-xylose, respectively. These monosaccharide-bound crystals were cryoprotected in a similar way as for the native crystal with solutions consisting of reservoir solution containing 35% (v/v) PEG 300 and the monosaccharide supplements. All cryoprotection and ligand soaking procedures were performed at 283 K.

The N. crassa WT strain and all deletion strains used in this work were obtained from the FGSC (http://www.fgsc.net, [75 (link)]; Additional file 2: Table S8). The strains were grown on 1× Vogel’s salts [76 ] and one of the following carbon sources (w/v): 1% arabinan (Megazyme, P-ARAB), 1% arabinogalactan (Megazyme, P-ARGAL), 2 mM l-arabinose (Sigma Aldrich, 10839), 1% cellulose (Avicel^® PH-101; Sigma Aldrich, 11365), 1% pectin (citrus peel, Sigma Aldrich, P9135), 1% polygalacturonic acid (PGA, Sigma Aldrich, 81325), 2 mM l-rhamnose (Sigma Aldrich, R3875), 2% sucrose (Sigma Aldrich, S7903), 1% xylan (beechwood, Sigma Aldrich, X4252), and 2 mM d-xylose (Sigma Aldrich, X3877). A medium containing 1× Vogel’s salts, 2 mM d-galacturonic acid (Sigma Aldrich, 48280), and 0.5 mM d-xylose was used for the growth experiment of N. crassa on d-galacturonic acid. The growth conditions are described further below.
The experiments regarding the pdr-1-oex strain were conducted on medium of 1× Vogel’s salts plus 1% (w/v) xylan. After 24 h of incubation at 25 °C, 200 rpm, and constant light, a solution containing 1× Vogel’s salts plus 0.5 mM l-Rha or d-GalA was added to the xylan medium two times a day for the remainder of the 3-day incubation period. Dry weight and endo-PGase activity were determined as explained in the respective method section.