1. Structural Biology and Molecular Biophysics
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Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer’s and Pick’s diseases

  1. Wenjuan Zhang
  2. Benjamin Falcon
  3. Alexey G Murzin
  4. Juan Fan
  5. R Anthony Crowther
  6. Michel Goedert  Is a corresponding author
  7. Sjors HW Scheres  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom
Research Article
Cite this article as: eLife 2019;8:e43584 doi: 10.7554/eLife.43584
7 figures, 2 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Different types of heparin-induced tau filaments.

(A) Cryo-EM image of heparin-induced 2N4R tau filaments. (B) Cryo-EM image of heparin-induced 2N3R tau filaments. 2D class averages of each filament type are shown as insets. Scale bars, 50 nm.

https://doi.org/10.7554/eLife.43584.002
Figure 1—figure supplement 1
Heparin-induced tau filaments can change from one type into another.

Scale bars, 50 nm.

https://doi.org/10.7554/eLife.43584.003
Figure 1—figure supplement 2
Cryo-EM image of heparin-induced filaments assembled from a mixture of 2N4R and 2N3R tau.

Scale bars, 50 nm.

https://doi.org/10.7554/eLife.43584.004
Figure 2 with 1 supplement
Cryo-EM structure of 2N4R tau snake filaments.

(A) β-strands and loop regions in the filaments are shown in different colours below the primary sequence of the microtubule-binding repeats (R1–R4). (B) Central slice of the 3D map. The position of the helical axis is indicated by a red cross, extra densities close to outward-facing lysines by yellow arrows, and extra density in front of the hydrophobic patches by a pink arrow. (C) Cryo-EM density with the atomic model. The sharpened, high-resolution map is in blue, and an unsharpened, 4.0 Å low-pass filtered map in grey. (D) Schematic view of the snake filament. (E) Rendered view of secondary structure elements in three successive rungs. (F) As in E, but in a view perpendicular to the helical axis.

https://doi.org/10.7554/eLife.43584.005
Figure 2—figure supplement 1
Fourier shell correlation curves and side views of the 3D reconstruction of 2N4R tau snake filaments.

(A) Fourier shell correlation curves between two independently refined half-maps (black, solid), of the final model versus the full map (red, solid), of a model refined in the first independent half map versus the first half map (green, solid), and of the same model versus the second independent half map, which was not used for refinement (blue, dashed) (B) Side views of the 3D reconstruction. The detailed view of the helical axis is at higher threshold to show it more clearly.

https://doi.org/10.7554/eLife.43584.006
Figure 3 with 1 supplement
Cryo-EM structure of 2N4R tau twister filaments.

(A) β-strands and loop regions in the filaments are shown in different colours below the primary sequence of the microtubule-binding repeats (R1–R4). (B) Central slice of the 3D map. The position of the helical axis is indicated by a red cross, extra densities close to the outward-facing lysines by yellow arrows, and extra density in front of the hydrophobic patches by pink arrows. (C) Cryo-EM density with the atomic model. The sharpened, high-resolution map is in blue, and an unsharpened, 4.0 Å low-pass filtered map in grey. (D) Schematic view of the twister filament. (E) Rendered view of secondary structure elements in three successive rungs. (F) As in E, but in a view perpendicular to the helical axis.

https://doi.org/10.7554/eLife.43584.007
Figure 3—figure supplement 1
Fourier shell correlation curves and side views of the 3D reconstruction of 2N4R tau twister filaments.

(A) Fourier shell correlation curves between two independently refined half-maps (black, solid), of the final model versus the full map (red, solid), of a model refined in the first independent half map versus the first half map (green, solid), and of the same model versus the second independent half map, which was not used for refinement (blue, dashed) (B) Side views of the 3D reconstruction. The detailed view of the helical axis is at higher threshold to show it more clearly.

https://doi.org/10.7554/eLife.43584.008
Figure 4 with 1 supplement
Cryo-EM structure of 2N4R tau jagged filaments.

(A) β-strands and loop regions in the filaments are shown in different colours below the primary sequence of the microtubule-binding repeats (R1–R4). (B) Central slice of the 3D map. The position of the helical axis is indicated by a red cross, extra densities close to the outward-facing lysines by yellow arrows, and extra density in front of hydrophobic patches by a pink arrow. (C) Cryo-EM density with the atomic model. The sharpened, high-resolution map is in blue, and an unsharpened, 4.0 Å low-pass filtered map in grey. (D) Schematic view of the jagged filament. (E) Rendered view of the secondary structure elements in three successive rungs. (F) As in E, but in a view perpendicular to the helical axis.

https://doi.org/10.7554/eLife.43584.009
Figure 4—figure supplement 1
Fourier shell correlation curves and side views of the 3D reconstruction of 2N4R tau jagged filaments.

(A) Fourier shell correlation curves between two independently refined half-maps (black, solid), of the final model versus the full map (red, solid), of a model refined in the first independent half map versus the first half map (green, solid), and of the same model versus the second independent half map, which was not used for refinement (blue, dashed) (B) Side views of the 3D reconstruction. The detailed view of the helical axis is at higher threshold to show it more clearly.

https://doi.org/10.7554/eLife.43584.010
Figure 5 with 1 supplement
Cryo-EM structure of 2N3R tau filaments.

(A) β-strands and loop regions in the filaments are shown in different colours below the primary sequence of the microtubule-binding repeats (R1–R4). (B) Central slice of the 3D map. The position of the helical axis is indicated by a red cross, extra densities close to outward-facing lysines by yellow arrows, and extra density in front of hydrophobic patches by pink arrows. (C) Cryo-EM density with the atomic model. The sharpened, high-resolution map is in blue, an unsharpened, 4.0 Å low-pass filtered map in grey. (D) Schematic view of 2N3R tau filament. (E) Rendered view of the secondary structure elements in three successive rungs. (F) As in E, but in a view perpendicular to the helical axis.

https://doi.org/10.7554/eLife.43584.012
Figure 5—figure supplement 1
Fourier shell correlation curves and side views of the 3D reconstruction of 2N3R tau filaments.

(A) Fourier shell correlation curves between two independently refined half-maps (black, solid), of the final model versus the full map (red, solid), of a model refined in the first independent half map versus the first half map (green, solid), and of the same model versus the second independent half map, which was not used for refinement (blue, dashed) (B) Side views of the 3D reconstruction. The detailed view of the helical axis is at higher threshold to show it more clearly.

https://doi.org/10.7554/eLife.43584.013
Figure 6 with 1 supplement
Immuno-EM of heparin-induced 2N4R and 2N3R tau filaments.

(A) Schematic of 2N4R tau with N-terminal inserts (N1 and N2) and microtubule-binding repeats (R1, R2, R3, R4) highlighted. The epitopes of antibodies BR133 (residues 1–16), BR136 (244-257), Anti4R (275-291), BR135 (323-335), TauC4 (354–369) and BR134 (428-441) are underlined. (B) Representative immuno-EM images with antibodies BR133, BR136, Anti4R, BR135, TauC4, and BR134 of heparin-induced 2N4R and 2N3R tau filaments without (-) and with pronase (+) treatment. Scale bar, 100 nm. (C) Table summarising the results from B, and comparison with the immuno-EM results of AD and PiD. Tick marks indicate antibody decoration of filaments; crosses indicate absence of decoration. The four boxes where the human diseases differ from the in vitro heparin-induced filaments are highlighted in blue.

https://doi.org/10.7554/eLife.43584.014
Figure 6—figure supplement 1
Western blots.

Western blots of recombinant 2N3R and 2N4R tau using BR133, BR136, Anti4R, BR135, TauC4 and BR134.

https://doi.org/10.7554/eLife.43584.015
Comparison of known tau filament structures.

(A) β-strands and loop regions in the filaments are shown in different colours below the primary sequence of the microtubule-binding repeats (R1–R4). (B) Schematic representation of the different tau folds: the paired helical filament (PHF) and straight filament (SF) from Alzheimer's disease (AD); the narrow Pick filament (NPF) and wide Pick filament (WPF) from Pick's disease (PiD); the heparin-induced 2N4R snake (4 R-s), twister (4 R-t) and jagged (4 R-j); and the 2N3R heparin-induced filaments (3R). (C) Comparison of the structures of heparin-induced filaments of 2N4R and 2N3R tau with those of tau protofilaments from AD and PiD.

https://doi.org/10.7554/eLife.43584.016

Tables

Table 1
Cryo-EM structure determination and model statistics
https://doi.org/10.7554/eLife.43584.011
4 R-s4 R-t4 R-j3R
Data collection and processing
MicroscopePolaraPolaraPolaraTitan Krios
Voltage (kV)300300300300
DetectorFalcon-IIIFalcon-IIIFalcon-IIIK2 (post-GIF)
Electron exposure (e–/Å2)50505050
Defocus range (μm)−1.7 to −2.8−1.7 to −2.8−1.7 to −2.8−0.8 to −2.2
Pixel size (Å)1.381.381.381.04
Initial particle images (no.)303,754187,55544,456788,359
Final particle images (no.)52,441141,46135,695149,909
Map resolution (Å)3.33.33.53.7
Helical rise (Å)4.704.704.704.70
Helical twist (°)−1.26−3.38−2.03−1.05
Refinement
Map sharpening B factor (Å2)−41.26−58.51−33.2−95.9
Model composition
Non-hydrogen atoms
Protein residues
13028468161218
177111105162
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.00940.01020.00990.0209
0.90071.07271.13421.0457
Validation
MolProbity score
Clashscore
Poor rotamers (%)
1.561.921.131.65
1.497.31.744.78
1.96000
Ramachandran plot
Favored (%)
Allowed (%)
Disallowed (%)
92.9890.9196.7794.0
10010010098.0
0002
EMPIAR10243102431024310242
EMDB4563456445654566
PDB6QJH6QJM6QJP6QJQ
Key resources table
Reagent type
(species)or resource
DesignationSource or referenceIdentifiersAdditional information
Recombinant DNAPlasmid:
pRK172-2N4R
PMID:
2124967; 8849730;
9407097
NCBI Reference
Sequence:
NM_005910.5
Plasmid can be
provided upon reasonable
request.
Recombinant DNAPlasmid:
pRK172-2N3R
PMID: 2124967NCBI Reference
Sequence:
NM_001203252.1
Plasmid can be provided
upon reasonable request.
Strain, strainback
ground (E. coli)
BL21 (DE3)Agilent Technologies200131
Chemical
compound, drug
HeparinSigma-AldrichH4784
Chemical
compound, drug
ChymostatinSigma-AldrichC7268Protease inhibitor
AntibodyBR133 (Anti-
N- terminus of tau
proteins, Rabbit
polyclonal)
In house
PMID: 28678775;
30158706
WB dilution: 1:4000
EM dilution: 1:50
AntibodyBR134 (Anti-
C- terminus of tau
proteins, Rabbit
polyclonal)
In house
PMID: 28678775;
30158706
WB dilution: 1:4000
EM dilution: 1:50
AntibodyBR136 (Anti-R1 of tau
proteins, Rabbit
polyclonal)
In house
PMID: 30158706;
30276465
WB dilution: 1:4000
EM dilution: 1:50
AntibodyAnti-4R (Anti-R2 of
2N4R tau protein,
Rabbit polyclonal)
Cosmo Bio
PMID: 28678775;
30158706; 30276465
CACTIP4RTP01WB dilution: 1:2000
EM dilution: 1:50
AntibodyBR135 (Anti-R3 of tau
proteins, Rabbit
polyclonal)
In house
PMID: 28678775;
30158706; 30276465
WB dilution: 1:4000
EM dilution: 1:50
AntibodyTauC4 (Anti-R4 of tau
proteins, Rabbit
polyclonal)
Masato Hasegawa
PMID: 28678775;
30158706; 30276465
WB dilution: 1:2000
EM dilution: 1:50
Software, algorithmRELIONPMID: 30412051RRID:SCR_016274
Software, algorithmCOOTPMID: 20383002RRID:SCR_014222
Software, algorithmREFMACPMID: 15299926RRID:SCR_014225
Software, algorithmPHENIXPMID: 20124702RRID:SCR_014224
  1. WB: Western Blot; EM: Electron microscopy.

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