Cryo-EM structure of heparin-induced 2N4R tau twister filaments
Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer's and Pick's diseases.
A stack of three consecutive monomers was refined to preserve nearest-neighbour interactions for the middle chain. Side-chain clashes were detected using MOLPROBITY, and corrected by iterative cycles of real-space refinement in COOT and Fourier-space refinement in REFMAC and PHENIX. For each refined structure, separate model refinements were performed against a single half-map, and the resulting model was compared to the other half-map to confirm the absence of overfitting.
we performed two rounds of 3D auto-refinement.
Aligned, non-dose-weighted micrographs were used to estimate the contrast transfer function (CTF) using CTFFIND4.1
Recombinant tau protein was induced into filaments by incubation with heparin at 37 C for 3 days
heparin-induced 2N4R tau twister filaments
Movie frames were gain-corrected, aligned, dose weighted and then summed into a single micrograph using MOTIONCOR2 (Zheng et al., 2017)
Images were collected in movie-mode at 30 frames per second
Recombinant tau protein was induced into filaments by incubation with heparin at 37 C for 3 days. The filaments were pronase-treated before making Cryo-grids.
Neurofibrillary tangle protein,Paired helical filament-tau,PHF-tau
Medical Research Council (United Kingdom)
We used Thioflavin T fluorescence to check the kinetic assays of the filament assembly.
We used negative-stain electron microscopy to check the morphology of assembled filaments.
Recombinant tau protein, heparin, filament, cross-beta structure, PROTEIN FIBRIL