Computational protocol: Structural bases for N-glycan processing by mannoside phosphorylase

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Protocol publication

[…] X-ray experiments were carried out at 100 K. Crystals of Uhgb_MP were soaked for a few seconds in reservoir solution supplemented with 15%(v/v) glycerol (apo) or 15%(v/v) PEG 300 (complexes) prior to flash-cooling. Apo Uhgb_MP diffraction data sets were collected on beamline ID23-1 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France, while those for the complexes were collected on the XALOC beamline at the ALBA Synchrotron, Cerdanyola del Vallès, Spain (Juanhuix et al., 2014). The diffraction intensities were integrated and scaled using XDS (Kabsch, 2010) and 5% of the scaled amplitudes were randomly selected and excluded from the refinement procedure. All crystals belonged to the ortho­rhombic space group P212121, with six molecules per asymmetric unit, giving Matthews coefficients of 2.22 and 2.11 Å3 Da−1 and solvent contents of 44 and 42% for the apo forms and the three complexes, respectively. The structures were solved by the molecular-replacement method using Phaser (McCoy et al., 2007) from the CCP4 software suite (Potterton et al., 2003) and chain A of the crystal structure of Tm1225 from T. maritima MSB8 (PDB entry 1vkd) as a search model for the apo form. The final translation-function Z-score was 42.8 and the R and R free values of the refined structure were 0.155 and 0.190, respectively. Once solved, the apo structure was then used to solve the protein–ligand structures. The structures of Uhgb_MP in complex with mannose, with N-acetylglucosamine and with mannose and N-acetyl­glucosamine were refined to final R/R free values of 0.150/0.193, 0.154/0.183 and 0.158/0.192, respectively, using REFMAC5 (Murshudov et al., 2011). Models were built manually in αA-weighted electron-density maps using Coot (Emsley & Cowtan, 2004). Water molecules were manually checked after automatic assignment and ligand molecules were manually fitted in residual maps. Refinement statistics are listed in Table 1. [...] Small-angle X-ray scattering (SAXS) experiments were performed on the SWING beamline at the SOLEIL synchrotron, Gif-sur-Yvette, France. The wavelength was set to 1.033 Å. A 17 × 17 cm Aviex CCD detector was positioned 1800 mm from the sample, with the direct beam off-centred. The resulting exploitable q-range was 0.006–0.6 Å−1, where q = 4πsinθ/λ, considering 2θ as the scattering angle. The samples were circulated in a thermostated quartz capillary with a diameter of 1.5 mm and 10 µm wall thickness positioned inside a vacuum chamber. A 80 µl volume of sample was injected onto a size-exclusion column (Bio SEC3 300, Agilent) equilibrated in phosphate-based buffer (20 mM potassium phosphate pH 7.0, 150 mM NaCl) or Tris-based buffer (20 mM Tris–HCl pH 7.0, 300 mM NaCl supplemented with 10% glycerol) using an Agilent high-performance liquid-chromatography (HPLC) system and eluted directly into the SAXS capillary cell at a flow rate of 200 µl min−1 at a temperature of 10°C. Samples were separated from the pushing liquid (water) by two air volumes of 6 µl each, as described previously (David & Pérez, 2009). SAXS data were collected online throughout the elution time and a total of 149 frames, each lasting 2 s, were recorded separated by a dead time of 0.5 s between frames. The transmitted intensity was continuously measured with an accuracy of 0.1% using a diode embedded in the beam stop. For each sample, the stability of the associated radius of gyration and the global curve shape in the frames corresponding to the main elution peak were checked, and the resulting selection of curves were averaged as described previously (David & Pérez, 2009). The recorded curves were normalized to the transmitted intensity and subsequently averaged using Foxtrot, a dedicated in-house application. The same protocol was applied to buffer scattering. R g values were determined by a Guinier fit of the one-dimensional curves using the ATSAS package (Petoukhov et al., 2007). The P(r) function was calculated using the GNOM program and the corresponding ab initio envelopes were calculated using the GASBOR program. Rigid-body SAXS modelling was performed using the CORAL program. […]

Pipeline specifications

Software tools XDS, CCP4, REFMAC5, Coot, GASBOR
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Mannose, Phosphates