To further evaluate the above conjecture, we generated the translocation pathway for SARS-CoV-2 RdRp with V557L mutation and performed MD simulations with the translocating intermediates (see SI Section 3 for details). We found that the distance between V557L and Remdesivir (5.5Å±0.4Å) is similar to that between V557L and wildtype adenosine (5.4Å±0.6Å) (Fig. and). In addition, Remdesivir and adenosine also form similar base stacking angles relative to the upstream nucleotide, with dihedral angles of 29.72°±6.42° and 30.34°±5.29°, respectively (Fig. and). Therefore, our results have suggested that the V557L mutation would rotate its side chain away from the translocation pathway and thereby relieve the hindrance over the translocation of Remdesivir from +2 to +1 site. Our calculations have rationalized the kinetic steady-state experimental data that the UTP incorporation opposite to Remdesivir is 5-fold more efficient with V557L mutation . Moreover, it further consolidates that the reduced UTP incorporation complementary to Remdesivir at +1 site is mainly attributed to the hampered translocation of Remdesivir from +2 to +1 site due to the steric repulsion from V557.