Cryo-EM analysis of PAD12 tau filaments

A Schematic of PAD12 tau. The amino-terminal inserts N1 (residues 44–73), and N2 (74–102) are shown in grey, the proline-rich region (151-243) is in light grey, the microtubule-binding repeats are in purple (R1, 244-274), blue (R2, 275-305), green (R3, 306-336) and yellow (R4, 337-368) and the carboxy-terminal domain (369-441) is shown in orange. The twelve phosphomimetic mutations of PAD12 are indicated with vertical lines. B Cryo-EM micrograph of PHFs composed of a 1:1 mixture of 0N3R and 0N4R PAD12 tau. Arrows show PHFs (blue) and single protofilaments with the Alzheimer tau fold (light blue) C Cross-sections of cryo-EM reconstructions perpendicular to the helical axis, with a thickness of approximately 4.7 Å for assembly reactions with 0N3R PAD12 tau, 0N4R PAD12 tau and a 1:1 mixture of 0N3R and 0N4R PAD12 tau. Inserts show pie charts with the particle distribution per filament types (PHFs in blue; single protofilaments with the Alzheimer fold in light blue; single protofilaments with the CTE fold in yellow; discarded solved particles (coloured) and discarded filaments in grey). D Cryo-EM density map of the 1:1 0N3R:0N4R PAD12 tau mixture in transparent grey, superimposed with its refined atomic model. E Backbone ribbon of the atomic model shown in D (blue), overlaid with the model of PHFs from AD brain (PDB-ID: 6hre) (grey).

cryo-EM statistics

Purification of 0N3R and 0N4R PAD12 tau

A Schematic of protein purification and assembly procedure. B Coomassie-stained SDS-PAGE (4-20%) after cation exchange chromatography of 0N3R PAD12 tau. B SDS-PAGE after size exclusion chromatography (SEC) of 0N3R PAD12 tau. C SEC profile of 0N3R PAD12 tau. D-F As A-C, but for 0N4R PAD12 tau.

Fourier shell correlation curves

A Fourier shell correlation curves between two independently refined half-maps (black); between the atomic model fitted in half-map 1 and half-map1 (blue); between that the atomic model fitted in half-map 1 and half-map 2 (yellow) and between the atomic model fitted in the sum of the two half-maps and the sum of the two half-maps (red), for the PAD12 PHF structure assembled from a 1:1 mixture of PAD12 0N3R and 0N4R tau (left) and from PAD12 0N3R tau, seeded with filaments that were extracted from the brain of an individual with AD (right).

Cryo-EM analysis of tau297-391 and PAD12 tau PHFs

A Cross-sections of cryo-EM reconstructions perpendicular to the helical axis, with an approximate thickness of 4.7 Å and pie charts showing the distribution of filament types (blue: PHFs; light blue: single protofilaments with the Alzheimer fold (not shown); grey: discarded filaments, for freeze-thawed filaments (flash frozen in liquid nitrogen, thawed at RT), for filaments assembled in an Eppendorf tube, for filaments assembled from PAD12 tau0-391, and for filaments assembled from PAD12 tau151-391. B-E Cryo-EM overviews of PHFs composed of residues tau297-391 (A), a 1:1 mixture 0N3R and 0N4R PAD12 tau (B), tau 0-391 (C) and 151-391 PAD12 3R tau (D). From left to right: grid square overview, foilhole overview, and acquisition image. Arrows indicate clumped aggregates of filaments (pink) and ice particles (blue).

EM analysis of 0N3R PAD12+4, PAD12-4 and PAD12+/-4 tau constructs

A Schematic of tau sequence as in Figure 1A, with the four extra mutations of PAD12+4 in blue and the four mutations that were removed from PAD12-4 in red. B Negative stain EM of filaments formed with PAD12+4 (left), PAD12-4 (middle) and PAD12+/-4 (right) 0N3R tau. C Cryo-EM reference-free 2D classes of filaments assembled from 0N3R PAD12-4. D Reference-free 2D classes of filaments assembled from 0N3R PAD12-4.

Labelling of pre-assembled PAD12 tau filaments

A Cartoon showing that filaments can be labelled via NHS-ester chemistry, which specifically targets primary amines in lysine residues. B SDS-PAGE 4-20% exposed at 488nm light. C Negative stain EM of DyLight488-labeled PAD12 3R tau filaments, with the Eppendorf tube of the corresponding, coloured protein pellet shown in the top right. The circular inset shows a cross-section of the corresponding cryo-EM reconstruction. D Immuno-EM showing biotinylated tau is labelled with streptavidin-coated 10 nm gold particles.

In vitro seeded assembly with PAD12 tau

A Schematic of experimental approach for the seeded assembly of PAD12 0N3R. Small amounts of brain material are used to seed the assembly of PAD12 0N3R (round 1). The filaments formed from round one are used as seeds for a second round. B ThT fluorescence profile of the AD-seeded (purple), second-round seeding (pink) and non-seeded control (yellow) N=9. The circles are individual measurements (normalised for each reaction). C Cross-sections of cryo-EM reconstructions perpendicular to the helical axis, with a thickness of approximately 4.7 Å and pie charts showing the distribution of filament types for the first (left) and second (right) round of seeding (pink/purple PHFs, yellow single protofilaments with the Alzheimer fold (not shown); grey discarded filaments). D Cryo-EM density map of AD-seeded 0N3R PAD12 tau (transparent grey) with the superimposed fitted atomic model. E Backbone ribbon of in vitro seeded PHF (purple) overlaid with AD PHF (grey; PDB-ID: 6hre).

Cellular seeding with recombinant tau filaments

A Box-plot showing the number of detected seeds, which were normalised to the number of cells and compared to mock-treated control cells (n ≥ 10,000 cells/condition analysed). Graph represents mean values; error bars represent standard deviation. B Images from control (-seed) and cells seeded with 0.25 µg of assembled tau297-391 PHFs, PAD12 0N3R PHFs and PAD12 0N3R:0N4R PHFs. Fixed cells were stained against HA for labelling over-expressed tau297-391 (green) and Hoechst (blue) for labelling of the nucleus. Scale bar, 50 µm. C Images from control cells without the addition of seeds (-seed) and cells seeded with 0.25 µg of PAD12 0N3R PHFs that were pre-labelled with DyLight-488. Fixed cells were stained against HA for labelling over-expressed tau (red) and Hoechst (blue) for labelling nuclei. Scale bar, 50 µm.

Cellular seeding with AD brain-derived tau and with PAD12 0N3R tau filaments labelled with DyLight-488 A,

B Box-plots showing the number of detected seeds which were normalised to the number of cells, and compared to mock-treated control cells (n ≥ 10,000 cells/condition analysed) for cells seeded with AD-brain-derived seeds (A) and PAD12 0N3R DyLight-488-labelled filaments (B). Graphs represent mean values; error bars represent standard deviations. C Images from control cells without the addition of seeds (-seed) and cells seeded with insoluble tau from 200 µg of sAD brain tissue or 0.25 µg PAD12 0N3R tau filaments that were pre-labelled with DyLight-488. Fixed cells were stained against HA for labelling overexpressed tau (green) and Hoechst dye (blue) for labelling nuclei. Scale bar, 50 µm.

NMR spectroscopy of wildtype and PAD12 tau297-441

A Secondary shift analysis of the backbone Cα and Cβ chemical shifts. Residues that have a preference for helical torsion angles have positive values, and those with an extended backbone preference have negative values. Secondary chemical shifts for tau297-391 are shown in grey, tau297-441 in black and PAD12 tau297-441 in lilac. B Chemical shift perturbation (CSP) map of the peak location differences between wildtype and PAD12 tau297-441. C Heteronuclear NOE (hetNOE) values for wildtype and PAD12 tau297-441 are shown in black and lilac, respectively. HetNOE values are sensitive to motion on the picosecond timescale. Residues within a permanent secondary structure element will have hetNOE values of ∼0.8; lower or negative values indicate increased backbone flexibility. D Lineshape differences in HSQC spectra as shown by normalised peak intensity. Values for tau297-391 are shown in grey, tau297-441 in black and PAD12 tau297-441 in lilac. Grey shading indicates the 297-391 region, the dashed box highlights residues that form the ordered core of the FIA and dashed lilac lines indicate mutated residues.

NMR spectroscopy of wildtype and PAD12 tau151-391

A Chemical shift perturbation (CSP) map of the peak location differences between wildtype and PAD12 tau151-391, with mutated residues shown in lilac. B Secondary chemical shift analysis of the backbone Cα and Cβ resonances. Residues that have a preference for helical torsion angles have positive values, and those with an extended backbone preference have negative values. Secondary chemical shifts for tau297-391 are shown in grey, wildtype tau151-391 in black and PAD12 tau151-391 in lilac. C Heteronuclear NOE (hetNOE) values for wildtype and PAD12 tau151-391 are shown in black and lilac, respectively. HetNOE values are sensitive to motion on the picosecond timescale. Residues within a permanent secondary structure element will have hetNOE values of ∼0.8; lower or negative values indicate increased backbone flexibility. D Lineshape differences in HSQC spectra as shown by normalised peak intensities. Values for tau297-391 are shown in grey, tau151-391 in black and PAD12 tau151-391 in lilac. Grey shading indicates the 297-391 region, the dashed box highlights residues that form the FIA and dashed lilac lines indicate mutated residues. Grey shading indicates the 297-391 region, the dashed box highlights residues that form the ordered core of the FIA and dashed lilac lines indicate mutated residues.

HSQC peak assignment of wildtype and PAD12 tau151-391

Assigned 700 MHz 15N–1H heteronuclear single quantum coherence (HSQC) spectrum of left, wildtype tau151-391 (black), and right PAD12 tau151-391 (lilac).

HSQC peak assignment of wildtype and PAD12 tau297-441

Assigned 700 MHz 15N–1H heteronuclear single quantum coherence (HSQC) spectrum of, from left to right, tau297-441 (black), overlaid and PAD12 tau297-441 (lilac).