1. Neuroscience
  2. Structural Biology and Molecular Biophysics

Assembly of recombinant tau into filaments identical to those of Alzheimer’s disease and chronic traumatic encephalopathy

  1. Sofia Lövestam
  2. Fujiet Adrian Koh
  3. Bart van Knippenberg
  4. Abhay Kotecha
  5. Alexey G Murzin
  6. Michel Goedert  Is a corresponding author
  7. Sjors HW Scheres  Is a corresponding author
  1. Medical Research Council Laboratory of Molecular Biology, United Kingdom
  2. Thermo Fisher Scientific, Netherlands
https://doi.org/10.7554/eLife.76494
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Cite this article
  1. Sofia Lövestam
  2. Fujiet Adrian Koh
  3. Bart van Knippenberg
  4. Abhay Kotecha
  5. Alexey G Murzin
  6. Michel Goedert
  7. Sjors HW Scheres
(2022)
Assembly of recombinant tau into filaments identical to those of Alzheimer’s disease and chronic traumatic encephalopathy
eLife 11:e76494.
https://doi.org/10.7554/eLife.76494
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4 figures, 3 tables and 3 additional files

Figures

Figure 1 with 8 supplements
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New electron cryo-microscopy (cryo-EM) structures.

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; residues 337–368 (R4) are shown in yellow; residues 369–441 (C-terminal domain) are shown in orange. The filament types (as defined in Table 1) are shown at the top left of each structure.

Figure 1—figure supplement 1
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Cross-sections of electron cryo-microscopy (cryo-EM) reconstructions.

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 (as defined in Table 1) is displayed in the bottom left, and the percentage of a given type in the cryo-EM data set is shown in the top right.

Figure 1—figure supplement 2
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Electron cryo-microscopy (cryo-EM) maps and models of new structures (part 1).

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 blue; residues 306–336 (R3) are shown in green; residues 337–368 (R4) are shown in yellow; residues 369–441 (C-terminal domain) are shown in orange. The filament types (as defined in Table 1) are shown at the top left of each structure.

Figure 1—figure supplement 3
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Electron cryo-microscopy (cryo-EM) maps and models of new structures (part 2).

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 blue; residues 306–336 (R3) are shown in green; residues 337–368 (R4) are shown in yellow; residues 369–441 (C-terminal domain) are shown in orange. The filament types (as defined in Table 1) are shown at the top left of each structure.

Figure 1—figure supplement 4
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Schematics of the tau folds (part 1).

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, and glycines in pink. PHF, paired helical filament; CTE, chronic traumatic encephalopathy.

Figure 1—figure supplement 5
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Schematics of the tau folds (part 2).

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, and glycines in pink.

Figure 1—figure supplement 6
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Fourier shell correlation curves (part 1).

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 in the first half-map against that half-map (blue); and for the refined atomic model in the first half-map against the second half-map (yellow). The resolutions where the black line drops below 0.143 and the red line drops below 0.5 are indicated. The corresponding filament type (as defined in Table 1) is shown at the top left of each graph. See Supplementary file 1 - Tables 1–25 for further details on data processing.

Figure 1—figure supplement 7
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Fourier shell correlation curves (part 2).
Figure 1—figure supplement 8
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Handedness of structure 41a.

(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 transparent) and atomic models are shown for the left- and right-handed options. (C) Close-up of carbonyls showing a better map-model fit for the left-handed map.

Figure 2 with 1 supplement
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Assembly of recombinant tau into filaments like Alzheimer’s disease paired helical filaments (AD PHFs).

(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 purple; R2 (275–305) is shown in blue; R3 (306–336) is shown in green; R4 (337–368) is shown in yellow; the C-terminal domain (369-441) is shown in orange. (B) Amino acid sequence of residues 244–441 of tau, with the same colour scheme as in A. (C–E:) Projected slices, with a thickness of approximately 4.7 Å, orthogonal to the helical axis for several cryo-microscopy (cryo-EM) reconstructions. The filament types (as defined in Table 1) are shown at the bottom left and the percentages of types for each cryo-EM data set are given at the top right of the images. (C) Conditions of Al-Hilaly et al., 2017, with shaking at 700 rpm. (D) Our adapted protocol, using 200 rpm shaking. From left to right; paired helical filament (PHF), triple helical filament, quadruple helical filament type 1, and quadruple helical filament type 2. (E) Our optimised conditions for in vitro assembly of relatively pure PHFs.( F) Cryo-EM density map (grey transparent) of in vitro assembled tau filaments of type 4a and the atomic model colour coded according as in A. (G) Backbone ribbon of in vitro PHF (grey) overlaid with AD PHF (blue).

Figure 2—figure supplement 1
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Electron cryo-microscopy (cryo-EM) micrographs and density maps comparing in vitro assembled paired helical filaments (PHFs) and Alzheimer’s disease paired helical filaments (AD PHFs).

(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, EMDB-14063) as top view (top) and side view (bottom). (C). Cryo-EM density maps of AD PHF (EMDB:0259) as top view (top) and side view (bottom). The black arrows point to densities in front of lysines 317 and 321; the green and pink arrows point to densities inside the C-shape.

Figure 3 with 3 supplements
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Assembly of recombinant tau into filaments like chronic traumatic encephalopathy (CTE) type II filaments.

(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 indicated in the bottom left. The percentages of types are shown in the top right of each panel. ( B) Cryo-EM density map (grey transparent) of filament type 8a and the corresponding atomic model with the same colour scheme as in Figure 1. (C–E) Backbone ribbon views of protofilament and filament folds. (C) In vitro NaCl filament type 8a (grey) overlaid with CTE type II (orange). (D) Extended and C-shaped protofilaments aligned at residues 338–354 for LiCl, filament types 9a and 9b (left) and NaCl, filament types 8a and 8b (right). (E) Filament types 8b (NaCl), 9a (LiCl), 10a (KCl), and 4a (MgCl2) aligned at residues 356–364. (F) Atomic view of residues 334–358. The distance between the Cα of L344 and I354 is indicated. Filament types 8a, 8b, 9a, 9b, and 10a are shown in light purple, dark purple, dark orange, light orange, and blue, respectively.

Figure 3—figure supplement 1
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Extended and C-shaped NaCl protofilaments.

(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 highlighted in A. (E) Cryo-microscopy density map (transparent grey) and the atomic model at residues 338–354. Filament types 8a and 8b are shown in dark and light orange, respectively.

Figure 3—figure supplement 2
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Extended and C-shaped LiCl protofilaments.

(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 highlighted in A. (E) Cryo-microscopy density map (transparent grey) and the atomic model at residues 338–354. Filament types 9a and 9b are shown in light and dark purple, respectively.

Figure 3—figure supplement 3
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Cryo-microscopy (cryo-EM) densities inside the cavities of KCl and NaCl filaments.

(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) Cryo-EM density at the tip of the protofilament of 8a with additional non-proteinaceous densities highlighted by arrows. Blue arrows represent putative water molecules; pink arrows represent putative K+ or Cl− ions. K+ ions are shown in pink; Cl− ions are shown in cyan. (B–D) Close-up view of putative K+ and Cl− ion pairs. Distances between K+ and Cl− ions, and Oγ of S341 or the carbonyl oxygen of G335 are indicated. (E) Cryo-EM density of the tip of the protofilament of 8a with putative Na+ and Cl− ions fitted into the density. Purple arrow represents putative Na+ or Cl− ions; Na+ ions are shown in purple; Cl− ions are shown in cyan. (F–G) Close-up view of putative Na+ and Cl− ions. Distances between the Na+ and Cl− ions and the Oγ of S341 are indicated. (H) Cryo-EM density of chronic traumatic encephalopathy type I (EMD:0527), with putative Na+ and Cl− ions modelled inside the additional density.

Figure 4 with 2 supplements
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The effects of protein length on tau filament assembly.

(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 from Alzheimer’s disease paired helical filaments (AD PHFs) were formed; a blue circle indicates that the AD protofilament fold was formed; a green tick indicates that AD PHFs were formed. Resulting filament types (as defined in Table 1) are indicated for each experiment. (B) Projected slices, with a thickness of approximately 4.7 Å, orthogonal to the helical axis for the filaments formed with the constructs in A. Filament types are indicated at the bottom left; percentages of filament types in each cryo-EM data set are shown in the top right. (*) 50:50 ratio of 266–391 (3R) and 297–391 (4R) tau.

Figure 4—figure supplement 1
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Comparison of in vitro assembled tau filaments with the globular glial tauopathy (GGT) fold.

(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) Close-up atomic view. 7p66 is shown in grey, 27a in green, 28a in pink, 29a in orange, 32a PF-1 in blue, and 32a PF-2 in purple.

Figure 4—figure supplement 2
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Coomassie stained sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel for constructs 297–391, 297–408, and 296–441 before and after filament assembly.

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.

Tables

Table 1
In vitro assembly conditions for all filament types.
Filament typesConstruct (residues)BufferShaking (rpm)Time(hr)Fold
1a297–39110 mM PB 10 mM DTT pH 7.470048New
2a–d297–39110 mM PB 10 mM DTT pH 7.420048AD
3a297–39110 mM PB 10 mM DTT pH 7.40.1 μg /ml dextran sulphate20048AD
4a297–39110 mM PB 10 mM DTT pH 7.4 200 mM MgCl220048AD
5a297–39110 mM PB 10 mM DTT pH 7.4 20 mM CaCl220048AD
6a–c266/297–391*10 mM PB 10 mM DTT pH 7.420048AD
7a–b266–273 –39110 mM PB 10 mM DTT pH 7.4 200 mM MgCl220048AD
8a–b266/297–39110 mM PB pH 7.4 10 mM DTT 200 mM NaCl20048CTE
9a–b266/297–39110 mM PB pH 7.4 10 mM DTT 200 mM LiCl20048New
10a–b266/297–39110 mM PB pH 7.4 10 mM DTT 200 mM KCl20048New
11a266/297–39110 mM PB pH 7.4 10 mM DTT 100 μM ZnCl220048New
12a266/297–39110 mM PB pH 7.4 10 mM DTT 200 μM CuCl220048New
13a266/297–39110 mM PB pH 7.4 10 mM DTT 20 mM MgCl2 50 mM KCl 50 mM NaCl20048New
14a–b266/297–39110 mM PB pH 7.4 10 mM DTT 20 mM MgCl2 100 mM NaCl20048New
15a–d266/297–39110 mM PB pH 7.4 10 mM DTT 10 mM MgSO4 100 mM NaCl20048CTE
16a–b266/297–39110 mM PB pH 7.4 10 mM DTT 10 mM NaHCO3 100 mM NaCl20048New
17a–c266/297–39110 mM PB pH 7.4 10 mM DTT 500 mM NaCl20048New
18a244–39150 mM PB pH 7.4 10 mM DTT 20 mM MgCl220076New
19a244–39150 mM PB pH 7.4 10 mM DTT 200 mM NaCl20076New
20a244–39110 mM PB 10 mM DTT 5 mM Na4P2O720076New
21a–b258–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New
22a266–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New
23a–c266–391PBS pH 7.4 10 mM DTT20048CTE
24a287–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD
25a300–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD
26a303–39110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD
27a305–37910 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New
28a297–42110 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New
29a297–41210 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New
30a297–40210 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New
31a297–39610 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048New
32a–b297–39410 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD
33a297–38410 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD
34a–b297–39410 mM PB pH 7.4 10 mM DTT70048AD
35a–d297–394PBS pH 7.4 10 mM DTT70048New
36a–c300–391PBS pH 7.4 10 mM DTT70048New
37a303–391PBS pH 7.4 10 mM DTT70048New
38a258–39110 mM PB pH 7.4 10 mM DTT70048GGT
39a–b258–39110 mM PB pH 7.4 10 mM DTT 5 mM phosphoglycerate70048GGT
40a258–39110 mM PB pH 7.4 10 mM DTT 300 ug/ul heparan sulphate70048GGT
41a258–39110 mM PB pH 7.4 10 mM DTT 0.1% NaN370048New
42a–b297–408 4-pmm*10 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD
43a297–441 4-pmm10 mM PB pH 7.4 10 mM DTT 200 mM MgCl220048AD
44a266–391 S356D10 mM PB pH 7.4 10 mM DTT 200 mM KCl20048New
45a266–391 S356D10 mM PB pH 7.4 10 mM DTT 200 mM NaCl20048New
46a0N4RPBS pH 7.4 5 mM TCEP 50 ug/mL polyA RNA20096New
47a0N4RPBS pH 7.4 5 mM TCEP 5 mM L-phosphoserine20096New

  1. 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.

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Recombinant DNA
reagent
Plasmid: pRK172-0N4RPMID:2124967NCBI ReferenceSequence:NM_005910.5All constructs
were derived
from this plasmid
Strain, strain
background
(Escherichia coli)		BL21(DE3)Agilent200,131Chemically
competent cells
Software,
algorithm		RELIONPMID:30412051RRID:SCR_016274RELION 4.0Helical reconstruction
Software,
algorithm		CootPMID:20383002RRID:SCR_014222Model building
Software,
algorithm		ISOLDEPMID:20383002Model refinement
Table 2
Data acquisition details for the different microscopes.
MicroscopeLMB Krios G1LMB Krios G2TFS GlaciosTFS Krios G4
Magnification105,00096,000165,000165,000
CameraK2*/K3Falcon 4Falcon 4Falcon 4
Energy filter (eV)20NA1010
Voltage (kV)300300200300
Electron exposure (e–/Å2)4030/404040
Defocus range (μm)1.5–31.2–2.50.6–1.20.6–1.2
Pixel size (Å)0.85/1.145*0.8240.6720.727

  1. LMB: Laboratory of Molecular Biology.

  2. *

    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.

Additional files

Supplementary file 1

Cryo-EM data processing, refinement and validation statistics (Supplementary Tables 1-25).

https://cdn.elifesciences.org/articles/76494/elife-76494-supp1-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/76494/elife-76494-transrepform1-v2.docx
Source data 1

Uncropped gel of Figure 4—figure supplement 2.

https://cdn.elifesciences.org/articles/76494/elife-76494-data1-v2.zip

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  1. Sofia Lövestam
  2. Fujiet Adrian Koh
  3. Bart van Knippenberg
  4. Abhay Kotecha
  5. Alexey G Murzin
  6. Michel Goedert
  7. Sjors HW Scheres
(2022)
Assembly of recombinant tau into filaments identical to those of Alzheimer’s disease and chronic traumatic encephalopathy
eLife 11:e76494.
https://doi.org/10.7554/eLife.76494