We next asked: how do AF2-based sampling strategies affect distograms?

We tested MinnieFold, AFsample2, and AF_cluster.

The biggest impact came from the AF2 version itself. Only AF2.3 mirrored AF3’s flexibility-aware distogram profile.

We run MD of apo and holo AK2 and revealed that holo AK2 hinge distance is sampled rarely in its apo state!

Strikingly, apo AF3 distogram reproduces the exact same behavior!!🤯

Surprisingly, AF3 apo and holo AK2 predictions beautifully reflect this conformational change, where C-ter of AK2 experiences a disorder-to-order transition upon beta-stacking itself to AIFM1👇

To track hinge motion, we concentrated on 230-233 CB–CB distances of AK2.

A primer on distograms: for each residue pair, AF outputs distance probability distributions.

Earlier, it was shown that the shape of the distogram peaks may inform us on protein flexibility.

How?
✔ Unimodal peak → rigid behavior
✔ Bimodal peak → flexible behavior

We want awe. We want discovery. And we want them now!

Huge thanks to Itai and Martin for the most enjoyable lecture on Night Science — a reminder that opennes lie at the heart of discovery!