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
Share this article
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
  2. Download BibTeX
  3. Download .RIS
4 figures, 3 tables and 3 additional files

Figures

Figure 1 with 8 supplements
Download asset Open asset
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; …

Figure 1—figure supplement 1
Download asset Open asset
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 …

Figure 1—figure supplement 2
Download asset Open asset
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 …

Figure 1—figure supplement 3
Download asset Open asset
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 …

Figure 1—figure supplement 4
Download asset Open asset
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, …

Figure 1—figure supplement 5
Download asset Open asset
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, …

Figure 1—figure supplement 6
Download asset Open asset
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 …

Figure 1—figure supplement 7
Download asset Open asset
Fourier shell correlation curves (part 2).
Figure 1—figure supplement 8
Download asset Open asset
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 …

Figure 2 with 1 supplement
Download asset Open asset
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 …

Figure 2—figure supplement 1
Download asset Open asset
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, …

Figure 3 with 3 supplements
Download asset Open asset
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 …

Figure 3—figure supplement 1
Download asset Open asset
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 …

Figure 3—figure supplement 2
Download asset Open asset
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 …

Figure 3—figure supplement 3
Download asset Open asset
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.( …

Figure 4 with 2 supplements
Download asset Open asset
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 …

Figure 4—figure supplement 1
Download asset Open asset
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) …

Figure 4—figure supplement 2
Download asset Open asset
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

Download links