ABSTRACT: The Nuclear Pore Complex (NPC) is the only passageway for macromolecules between nucleus and cytoplasm, and one of localization microscopy's most important reference standards: it is massive and stereotypically arranged. The average architecture of NPC proteins has been resolved with pseudo-atomic precision, however observed NPC heterogeneities such as varying diameters, elongated shapes, deviant symmetries, and irregular shapes evidence a high degree of divergence from this average. Single Molecule Localization Microscopy (SMLM) images NPCs at protein-level resolution, whereupon image analysis software studies NPC variability. However the true picture of NPC variability is not known. In quantitative image analysis experiments, it is thus difficult to distinguish intrinsically high SMLM noise from true variability of the underlying structure.
We simulate structurally variable synthetic NPCs based on architectural models of the true NPC. We represent the NPC as a spring-model such that arbitrary deforming forces simulate irregularly shaped variations. Here, we provide datasets of simulated human NPCs, with multiple types of geometric variability and increasing magnitudes of deforming forces. Accompanying ground truth annotations allow to test the capabilities of image analysis software and facilitate a side-by-side comparison with real data. Photophysics are simulated using SMAP (Ries 2020, https://doi.org/10.1038/s41592-020-0938-1)
Simulation 1:
N-terminally labelled Nup107 from PDB model 5A9Q (DOI: 10.2210/pdb5A9Q/pdb)
For Nup107, we simulated eight classes representing commonly observed or hypothesized variability: unmodified reference NPCs ("standard"), smaller radius ("narrow"), smaller nucleoplasmic-cytoplasmic ring distance ("short"), elongated NPCs ("elongated"), Variant azimuthal angle between the nucleoplasmic and cytoplasmic ring("twisted"), 9-fold symmetry ("ninefold"), shifted nucleoplasmic and cytoplasmic ring ("shifted"), and independently tilted nucleoplasmic and cytoplasmic ring (tilted").
Simulation 2:
C-terminally labelled Nup96 from PDB model 5A9Q (DOI: 10.2210/pdb5A9Q/pdb)
We sweep two parameters that contribute to the variability of NPC radii: The standard deviation of the NPC radius, and the magnitude of irregular variability of NPCs. Irregular variability contributes to geometric variability, such as ellipticity, ring-tilt, ring-distance, and ring-shift, as well as to true irregular variability, measured as the residual sum of squares from fitted ellipses. We simulate each 1000 NPCs with a radius sampled with a standard deviation of 0, 1, and 2 nm; as well as irregular variability of 1, 10, and 20, resulting in 9 combinations of radius variability and irregular variability.
