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Detailed understanding of neuraminidase catalysis could allow to model and predict the effect of various mutations on enzyme’s substrate specificity and design its effective inhibitors. This issue is especially critical regarding the highly pathogenic avian influenza viruses (HPAIV), including H5N1 strain, causing disease outbreaks not only in the bird population, but also in humans (WHO data [1]). Viral neuraminidase selectively hydrolyzes sialylated glycans, being one of the key HPAIV elements ensuring receptor specificity for a particular strain which varies significantly be-tween avian and human populations thus providing a natural barrier for direct transmission. The study of H5N1 neuraminidase reactivity towards human receptors is paramount since the exact scheme of the mechanism and the evaluation of the catalytic stages are still lacking. The interaction of influenza neuraminidase with oligosaccharide fragments of sialylated gly-cans is a complex process determined by the multidimensional phase space of various confor-mations [2]. Thus, we have used comprehensive analysis of glycan’s conformational phase space to avoid unsystematic interpretation of the particular conditions of modeling as in one of the studies [3] authors failed to obtain an adequate model of the substrate specificity of H5N1 neuraminidase known to possess higher activity toward avian receptors than human ones comparing to seasonal strains [4]. The study has been performed using 6’sialolactose (α-Neu5Ac-2,6-β-Gal-1,4-β-Glc-O-Me) as a carbohydrate fragment of the receptor. Structure of a Michaelis complex has been revealed by using nonparametric Bayesian clustering of glycan structures obtained from 500 ns molecular dynamics simulation on the MSU Lomonosov 2 supercomputer [5]: most populated cluster permit-ting the distortion of sialic acid ring structure has been taken into account. Formation of a covalent glycosyl-enzyme complex has been evaluated applying multidimensional free energy calculation by combined quantum-mechanical/molecular-mechanical metadynamics. The results, together with our previously determined free energy profile for 3’sialolactose hydrolysis demonstrate how differences in the chemical structure of oligosacharides affect catalytic efficiency and convenient for evaluating adaptive mutations that evolve human tropism. This study was supported by the RFBR grant No 18-34-00953.