Rigidity analysis of Vaccinia Virus D13. The asymmetric unit of PDB file 3SAQ (a) is composed of two chains, A and B. We analyzed the rigidity of just chain A (b), just chain B (c), the biological assembly made up of three copies of chain A (g), and the biological assembly made up of three copies of chain B (f). Due to the rotamer nature of certain amino acids, hydrogen atoms were placed by the Reduce software at different rotamer positions for certain residues, including residue 511, Threonine (residue 511 for subunits 1 and 2 are show in (d) and (e)). This caused subunit 1 to have a different number of hydrogen bonds than subunits 2 and 3, resulting in the non-symmetric rigidity of the second biological assembly. When KINARI's curation tools were used to adjust for this discrepancy of hydrogen bonds, the resulting biological assembly was symmetrically rigid (h), as expected.