Structural dynamics of the nuclear pore complex

•High-speed atomic force microscopy (HS-AFM) captures movies of native nuclear pore complexes (NPCs) at transport-relevant millisecond timescales.•This reveals the NPC transport barrier for the first time, which consists of highly dynamic FG Nups that fluctuate rapidly inside the central channel.•Dynamic sub-structures such as the cytoplasmic filaments and the nuclear basket may play a role in facilitating the translocation of large cargoes through the NPC. Nuclear pore complexes (NPCs) are the sole conduits that facilitate macromolecular exchange between the nucleus and cytosol. Recent advancements have led to a more highly resolved NPC structure. However, our understanding of the NPC modus operandi that facilitates transport selectivity, and speed, of diverse cargoes remains incomplete. For the most part, assorted cargo-complexes of different sizes traverse the NPC central channel in milliseconds, yet little is known about the nanoscopic movements of its barrier-forming Phe-Gly nucleoporins (FG Nups) and related sub-structures at transport-relevant time and length scales. Here, we discuss how dynamic FG Nup behavior may confer NPCs with an effective permeability barrier according to the functional needs of the cell. Moreover, we postulate that structural flexibility might resonate throughout the NPC framework from the cytoplasmic filaments to the nuclear basket.