An integrative protocol for the structure determination of the mouse ASC-PYD filament.

Several inflammasomes that activate as part of the eukaryotic innate immune response contain long helical filaments of the adaptor protein ASC as a central structural element. Here, we describe a technical protocol that has enabled the structure determination of the filament of the ASC pyrin domain. The protocol integrates data from cryo-electron microscopy and solid-state NMR spectroscopy into a single simulated annealing protocol to determine structural coordinates that fit all input data optimally. The structure shows that the ASC pyrin domain filament is formed by helical stacking of individual pyrin domains forms and that the CARD domains are flexibly attached to the filament outside. An artificial perturbation of the input data shows that the integrated structure determination protocol can allow high quality structures even at resolutions of the electron density map as low 8Å. The protocol is extendable to other structural input data from biochemical or biophysical experiments.

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