en transferred into a cryo-protection solution and flash-frozen in liquid nitrogen. Data collection and structure determination All diffraction data were processed with HKL2000. The structure of DrPOMK was determined by single-wavelength dispersion method using data collected from a Se-Met crystal. Heavy atom search, phase calculation and refinement, density modification, and initial model building were carried out with Phenix. The structural model was manually traced in Coot. The transition state complex structure was determined by molecular replacement using the Se-Met structure in Phaser and refined using Phenix. nger et al., 1998). Five percent randomly selected reflections were used for cross-validation. Structural alignment between POMK and PKA was performed using Dali. Interaction between GalNAc-b3-GlcNAc-b 4-Man and POMK was analyzed using PISA. The structural model of human POMK was generated using Swiss-Model. Molecular graphics were prepared using PyMol. NMR spectroscopy 1D 1H NMR spectra of the trisaccharide GGM-MU in the absence and presence of DrPOMK were acquired at 25C on a Bruker Avance II 800 MHz NMR spectrometer buy UPF 1069 equipped with a cryoprobe Zhu et al. eLife 2016;5:e22238. DOI: 10.7554/eLife.22238 14 of 18 Research article Biochemistry Biophysics and Structural Biology using a 50 ms T2 filter consisting of a train of spin-lock pulses to eliminate the broad resonances from the protein. DrPOMK titrations were performed in 25 mM Tris, 180 mM NaCl, and 10 mM MgCl2 in 98% D2O. The 13C and 1H resonances of the trisaccharide were reported previously and confirmed in the current study. The 1 H chemical shifts are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate. The NMR spectra were processed using NMRPipe and analyzed using NMRView. The glycan binding affinity to DrPOMK was determined using glycan-observed NMR experiments as described recently. For the resolved anomeric trisaccharide peak, the bound fraction was calculated by measuring the difference in the peak intensity in the absence and presence of DrPOMK, and then dividing by the peak intensity of the free form. To obtain dissociation constant, the data were fitted to the standard quadratic equation using GraphPad Prism. The standard deviation from data fitting is reported. Acknowledgements We are grateful to staff members of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826959 Advanced Light Source and Shanghai Synchrotron 
Radiation Facility for assistance in X-ray data collection. We thank Hiroshi Suga of Hiroshima Prefectural University for the culture of Capsaspora, Shitang Huang of the Core Facilities at School of Life Sciences, Peking University for help with experiments involving ATP, and Allison Bouska for assistance with DNA preparation. We thank Carolyn Worby, Alexandr Kornev, Susan Taylor, Erhard Hohenester, Jeffrey Esko, and members of the Xiao laboratory for insightful discussions and comments regarding the manuscript. Additional information Funding Funder Grant reference number Author Jack E Dixon National Institute of Diabetes DK18849 and Digestive and Kidney Diseases National Institute of Diabetes DK18024 and Digestive and Kidney Diseases Howard Hughes Medical Institute Paul D. Wellstone Muscular Dystrophy Cooperative Research Center National Natural Science Foundation of China National Key Research and Development Plan of China 1U54NS053672 Jack E Dixon Kevin P Campbell Kevin P Campbell 31570735 2016YFC0906000 Junyu Xiao Junyu Xiao The funders had no role in study design, data collection and interpret
