Backbone traces for filaments with previously unobserved structures. Residues 244–274 (R1) are shown in purple; residues 275–305 (R2) are shown in blue; residues 306–336 (R3) are shown in green; …
Projected slices perpendicular to the helical axis, and with a thickness of approximately 4.7 Å, are shown for all cryo-EM structures described in this paper. For each structure, the filament type …
Cryo-EM density maps (grey transparent) and atomic models are shown for filaments with previously unobserved structures. Residues 244–274 (R1) are shown in purple; residues 275–305 (R2) are shown in …
Cryo-EM density maps (grey transparent) and atomic models are shown for filaments with previously unobserved structures. Residues 244–274 (R1) are shown in purple; residues 275–305 (R2) are shown in …
Negatively charged residues are shown in red, positively charged residues in blue, polar residues in green, non-polar residues in white, sulphur-containing residues in yellow, prolines in purple, …
Negatively charged residues are shown in red, positively charged residues in blue, polar residues in green, non-polar residues in white, sulphur-containing residues in yellow, prolines in purple, …
FSC curves are shown for two independently refined cryo-microscopy (cryo-EM) half-maps (black); for the final refined atomic model against the final cryo-EM map (red); for the atomic model refined …
(A) Fourier shell correlation curves for the final refined atomic model against the left-handed map (green) and against the right-handed map (orange). (B) Cryo-microscopy density maps (grey …
(A) Schematic of 2N4R tau sequence with domains highlighted. The regions 1N (44–73), 2N (74–102), P1 (151–197), and P2 (198–243) are shown in increasingly lighter greys; R1 (244–274) is shown in …
(A) Cryo-EM micrographs with filaments from in vitro assembly conditions 1 (left), 2 (middle), and 4 (right). Scale bar represents 100 Å.( B) Cryo-EM density maps of in vitro PHF (filament type 4a, …
(A) Projected slices, with a thickness of approximately 4.7 Å, orthogonal to the helical axis are shown for different assembly conditions and filament types (as defined in Table 1), which are …
(A) Backbone ribbon view of C-shaped (filament type 8a) and extended (filament type 8b) protofilaments formed with NaCl, aligned at residues 338–354. (B–D) Close-up atomic view of regions …
(A) Backbone ribbon view of extended (filament type 9a) and C-shaped (filament type 9b) protofilaments formed with LiCl, aligned at residues 338–354.( B–D) Close-up atomic view of regions …
(A–H) Cryo-EM density map (transparent grey) and the corresponding atomic models for filament types 7a (assembled with NaCl) and 8a (assembled with KCl) are shown in orange and green, respectively.( …
(A) Schematic representation of 2N4R tau and the constructs used in this study. A red cross indicates that no filaments were formed; an orange dash indicates that filaments with structures distinct …
(A) Backbone ribbon view of GGT type 1 (PDB:7p66) and tau filaments from in vitro assembled filament types 27a, 28a, 29a, 32a protofilament (PF)1, and 32a PF2, aligned at residues 288–322. (B) …
About 3.5 μg of protein was loaded into each well. f: after filament assembly; *: constructs with the four-phospho-mimetic mutations: S396D, S400D, T403D, and S404D.
Filament types | Construct (residues) | Buffer | Shaking (rpm) | Time(hr) | Fold |
---|---|---|---|---|---|
1a | 297–391 | 10 mM PB 10 mM DTT pH 7.4 | 700 | 48 | New |
2a–d | 297–391 | 10 mM PB 10 mM DTT pH 7.4 | 200 | 48 | AD |
3a | 297–391 | 10 mM PB 10 mM DTT pH 7.40.1 μg /ml dextran sulphate | 200 | 48 | AD |
4a | 297–391 | 10 mM PB 10 mM DTT pH 7.4 200 mM MgCl2 | 200 | 48 | AD |
5a | 297–391 | 10 mM PB 10 mM DTT pH 7.4 20 mM CaCl2 | 200 | 48 | AD |
6a–c | 266/297–391* | 10 mM PB 10 mM DTT pH 7.4 | 200 | 48 | AD |
7a–b | 266–273 –391 | 10 mM PB 10 mM DTT pH 7.4 200 mM MgCl2 | 200 | 48 | AD |
8a–b | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 200 mM NaCl | 200 | 48 | CTE |
9a–b | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 200 mM LiCl | 200 | 48 | New |
10a–b | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 200 mM KCl | 200 | 48 | New |
11a | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 100 μM ZnCl2 | 200 | 48 | New |
12a | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 200 μM CuCl2 | 200 | 48 | New |
13a | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 20 mM MgCl2 50 mM KCl 50 mM NaCl | 200 | 48 | New |
14a–b | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 20 mM MgCl2 100 mM NaCl | 200 | 48 | New |
15a–d | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 10 mM MgSO4 100 mM NaCl | 200 | 48 | CTE |
16a–b | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 10 mM NaHCO3 100 mM NaCl | 200 | 48 | New |
17a–c | 266/297–391 | 10 mM PB pH 7.4 10 mM DTT 500 mM NaCl | 200 | 48 | New |
18a | 244–391 | 50 mM PB pH 7.4 10 mM DTT 20 mM MgCl2 | 200 | 76 | New |
19a | 244–391 | 50 mM PB pH 7.4 10 mM DTT 200 mM NaCl | 200 | 76 | New |
20a | 244–391 | 10 mM PB 10 mM DTT 5 mM Na4P2O7 | 200 | 76 | New |
21a–b | 258–391 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | New |
22a | 266–391 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | New |
23a–c | 266–391 | PBS pH 7.4 10 mM DTT | 200 | 48 | CTE |
24a | 287–391 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | AD |
25a | 300–391 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | AD |
26a | 303–391 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | AD |
27a | 305–379 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | New |
28a | 297–421 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | New |
29a | 297–412 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | New |
30a | 297–402 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | New |
31a | 297–396 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | New |
32a–b | 297–394 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | AD |
33a | 297–384 | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | AD |
34a–b | 297–394 | 10 mM PB pH 7.4 10 mM DTT | 700 | 48 | AD |
35a–d | 297–394 | PBS pH 7.4 10 mM DTT | 700 | 48 | New |
36a–c | 300–391 | PBS pH 7.4 10 mM DTT | 700 | 48 | New |
37a | 303–391 | PBS pH 7.4 10 mM DTT | 700 | 48 | New |
38a | 258–391 | 10 mM PB pH 7.4 10 mM DTT | 700 | 48 | GGT |
39a–b | 258–391 | 10 mM PB pH 7.4 10 mM DTT 5 mM phosphoglycerate | 700 | 48 | GGT |
40a | 258–391 | 10 mM PB pH 7.4 10 mM DTT 300 ug/ul heparan sulphate | 700 | 48 | GGT |
41a | 258–391 | 10 mM PB pH 7.4 10 mM DTT 0.1% NaN3 | 700 | 48 | New |
42a–b | 297–408 4-pmm* | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | AD |
43a | 297–441 4-pmm | 10 mM PB pH 7.4 10 mM DTT 200 mM MgCl2 | 200 | 48 | AD |
44a | 266–391 S356D | 10 mM PB pH 7.4 10 mM DTT 200 mM KCl | 200 | 48 | New |
45a | 266–391 S356D | 10 mM PB pH 7.4 10 mM DTT 200 mM NaCl | 200 | 48 | New |
46a | 0N4R | PBS pH 7.4 5 mM TCEP 50 ug/mL polyA RNA | 200 | 96 | New |
47a | 0N4R | PBS pH 7.4 5 mM TCEP 5 mM L-phosphoserine | 200 | 96 | New |
DTT: 1,4-dithiothreitol PB: Na2HPO4, NaH2PO4; Dextran sulphate: molecular weight 7–20 kDa (9011-18-1, Sigma-Aldrich); Heparan sulphate: 50–200 disaccharide units (57459-72-0, Sigma-Aldrich); Poly-A RNA (26763-19-9, Sigma-Aldrich); PBS: Phospho-buffered saline; TCEP: Tris(2-carboxyethyl) phosphine; *4-pmm: four-phospho mimetic mutations: S396D S400D T403D S404D *PB: Na2HPO4, NaH2PO4;*266/297–391: 50:50 ratio of 266LKHQ269 (3 R) and 297IKHV300 (4 R) –391. Fold: AD: Alzheimer’s Disease protofilament fold, CTE: chronic traumatic encephalopathy protofilament fold and GGT: globular glial tauopathy-like fold, New: new tau fold.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Recombinant DNA reagent | Plasmid: pRK172-0N4R | PMID:2124967 | NCBI ReferenceSequence:NM_005910.5 | All constructs were derived from this plasmid |
Strain, strain background (Escherichia coli) | BL21(DE3) | Agilent | 200,131 | Chemically competent cells |
Software, algorithm | RELION | PMID:30412051 | RRID:SCR_016274 | RELION 4.0Helical reconstruction |
Software, algorithm | Coot | PMID:20383002 | RRID:SCR_014222 | Model building |
Software, algorithm | ISOLDE | PMID:20383002 | Model refinement |
Microscope | LMB Krios G1 | LMB Krios G2 | TFS Glacios | TFS Krios G4 |
---|---|---|---|---|
Magnification | 105,000 | 96,000 | 165,000 | 165,000 |
Camera | K2*/K3 | Falcon 4 | Falcon 4 | Falcon 4 |
Energy filter (eV) | 20 | NA | 10 | 10 |
Voltage (kV) | 300 | 300 | 200 | 300 |
Electron exposure (e–/Å2) | 40 | 30/40 | 40 | 40 |
Defocus range (μm) | 1.5–3 | 1.2–2.5 | 0.6–1.2 | 0.6–1.2 |
Pixel size (Å) | 0.85/1.145* | 0.824 | 0.672 | 0.727 |
LMB: Laboratory of Molecular Biology.
LMB Krios G1 initially had a K2 camera, which was later replaced by a K3 camera. Only condition 1 a (as defined in Table 1) was collected on the K2 camera. The pixel size on the K2 camera was 1.145 Å.
Cryo-EM data processing, refinement and validation statistics (Supplementary Tables 1-25).
Uncropped gel of Figure 4—figure supplement 2.