For comparative purposes, we performed KIN analysis on both static (crystallographic) structures available in the Protein Data Bank (4) and on molecular dynamics trajectories of the respective structures. MD simulations of all 69 structures were performed and prepared according to the protocol described in the Supplementary Methods. A tunable cutoff parameter specifies what fraction of the simulation frames an interaction must be present in order to be counted for that protein. Figure shows the dependence of interactions detected on cutoff value, compared to analysis of PDB structures only. We will refer to this reference analysis of PDB structures as the crystallographic network. As might be expected, including all interactions present in any frames (cutoff >0%) of the simulation is highly sensitive but skewed by rare events. Setting the cutoff to even ≥10% still captures almost all of the crystallographic network while reducing the number of transient interactions (Figure ). Such transient interactions may be functionally important or may be artefactual, which is why a user-tunable parameter is critical here. Increasing the cutoff values between 10 and 50% results in a network more similar to the crystallographic network, with the number of shared residues between the two networks decreasing a lot slower than the number of the MD contacts (Figure ). This means that the interactions removed as a result of the increasing cutoff are largely non-crystallographic. A visual comparison of the conserved interaction network obtained from MD simulations using per-trajectory cutoffs is rendered in Figure . The majority of interactions that are preserved between crystallographic and MD-based networks (blue) describe interactions within secondary structure components. As might be expected, interactions found only via MD analysis are located primarily in the more flexible regions of the system. It is important to note that many of the dynamic interactions that are discarded when using a higher cutoff are strongly conserved across class A ß-lactamases and thus may contain important information (Figure ). For example, we observe that, of the interactions preserved when a per-trajectory cutoff of 10% is used instead of 50%, 32% (47 out of 143) are conserved in >80% of ß-lactamase proteins analyzed. Further, while a number of hydrogen bonding interactions dominate both MD-based and crystallographic networks, cutoff sensitivity analysis shows many hydrophobic interactions to generally be more transient (Figure ). Many of these interactions are not present in the crystallographic network and yet are highly conserved across the family.