To further examine if the presence of Remdesivir would affect the translocation dynamics, a translocation pathway was generated by the Climber algorithm and MD simulations were conducted for these translocating intermediates (see SI Section 3 for details). Intriguingly, we found that the 1'-cyano group of Remdesivir would clash with the side chain of V557 when it moves from +2 to +1 site (Fig. ), while such repulsion from V557 has not been observed when adenosine translocates. Specifically, when Remdesivir translocates from +2 to +1 site, its steric clash with V557 would result in a larger separation distance of 5.87Å±0.25Å compared to that for adenosine (4.91Å±0.12Å) (Fig. and). Moreover, the steric clash would push the base of Remdesivir in a tilted configuration with a larger dihedral angle relative to the base of the upstream nucleotide (55.95°±5.81°), while the adenosine's base adopts a more parallel configuration (25.04°±1.43°) (Fig. ). Therefore, our simulations have suggested that V557 hinders the translocation of Remdesivir from +2 to +1 site and further inhibit the NTP incorporation against Remdesivir at +1 site. This finding is also consistent with the experimental observation that SARS-CoV-2 RdRp with single-point V557L mutation would become drug resistant to Remdesivir 17 .