1. Neuroscience
  2. Structural Biology and Molecular Biophysics
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Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy

  1. Ricardo Guerrero-Ferreira
  2. Nicholas MI Taylor
  3. Ana-Andreea Arteni
  4. Pratibha Kumari
  5. Daniel Mona
  6. Philippe Ringler
  7. Markus Britschgi
  8. Matthias E Lauer
  9. Ali Makky
  10. Joeri Verasdonck
  11. Roland Riek
  12. Ronald Melki
  13. Beat H Meier
  14. Anja Böckmann  Is a corresponding author
  15. Luc Bousset  Is a corresponding author
  16. Henning Stahlberg  Is a corresponding author
  1. University of Basel, Switzerland
  2. University of Copenhagen, Denmark
  3. Institut de Biologie Intégrative de la Cellule (I2BC), CEA, CNRS, Université Paris Sud, Université Paris-Saclay, France
  4. Institut Fancois Jacob (MIRCen), CEA and Laboratory of Neurodegenerative Diseases, CNRS, France
  5. ETH Zurich, Switzerland
  6. Roche Innovation Center Basel, Switzerland
  7. Institut Galien Paris-Sud, CNRS, Université Paris-Sud, Université Paris-Saclay, France
  8. Labex Ecofect, UMR 5086 CNRS, Université de Lyon, France
Research Advance
Cite this article as: eLife 2019;8:e48907 doi: 10.7554/eLife.48907
4 figures, 2 videos, 4 tables, 5 data sets and 1 additional file

Figures

Figure 1 with 3 supplements
Cross-sections of the α-Syn polymorph 2a and 2b cryo-EM structures.

(A) Sequence of human α-Syn with familial PD mutation sites indicated in red. ß strands are indicated by arrows colored from blue to orange. Cryo-EM densities and atomic models of polymorph 2a (B) and polymorph 2b (C) of α-Syn. Each cryo-EM map shows two protofilaments (blue and orange) forming a fibril. PD-associated mutations sites, and first and last residues of the NAC regions are indicated. (D and E) Rainbow rendering views of the secondary structure elements in five successive rungs of both polymorphs. A view perpendicular to the helical axis is shown to illustrate the height differences in a single α-Syn fibril. Colors correspond to the arrows in the sequence displayed in panel (A).

Figure 1—figure supplement 1
Local resolution estimation and FSC curves.

Cryo EM maps with local resolution estimations for α-Syn polymorph 2a (A) and α-Syn polymorph 2b (F). The color scales indicate the resolution values within each map. (B) and (J) Fourier shell correlation curves between two independently refined half-maps. Overall resolutions are indicated. (C and G) Reference-free 2D class averages with their power spectra. (D) and (H) 2D projections of the cryo-EM maps with their power spectra. (E and I) 2D projection of the atomic models with their power spectra. White arrows in spectrum panels indicate the layer line at 1/4.8 Å with either a meridional n = 0 Bessel function peak (α-Syn polymorph 2a, panels (C) (D) and (E)), or off-meridional n = 1 Bessel function peak intensities (α-Syn polymorph 2b, panels (G) (H) and (I)).

Figure 1—figure supplement 2
Interface regions between two protofilaments of the α-Syn polymorph 2a and 2b.

(A and B) Views along the axis of the α-Syn polymorph 2a and 2b fibrils, respectively. The area shown is highlighted with squares in the ribbon diagrams (inserts). (C and D) Side views of the fibrils showing the side-by-side alignment of α-Syn molecules in polymorph 2a (C), compared to the staggered packing of α-Syn molecules in polymorph 2b (D). Viewing directions in (C) and (D) are indicated by arrows in the ribbon diagrams.

Figure 1—figure supplement 3
NMR identification of the residues forming the N-terminal beta strand.

(A) NMR secondary chemical shift plot according to reference (Gath et al., 2014b). β-strands are revealed when three or more residues in a row show a negative difference of the secondary shifts Δδ (the difference between the chemical-shift deviations of the observed Cα and Cβ shifts from the random coil shift). This information is plotted on the cryo-EM structure in red. (B) Extract of a 2D 13C-13C PAR spectrum (full aliphatic region shown in (D) of the cross peaks assigned to S87-A17, A18, A19 interactions, for which all assignment possibilities within 0.15 ppm are indicated. (C) Structural restraints resulting from the cross signals in (B), and from the A19S87 peak highlighted by a box in (D), where the full 2D 13C-13C PAR NMR spectrum is shown with assigned peaks corresponding to at least i, i ± 3 contacts (non-trivial structural restraints). Most of these peaks are ambiguous, that is, they have several assignment possibilities within 0.15 ppm, which were lifted here by comparison with the cryo-EM structures. This illustrates that the PAR spectrum indeed contains numerous structurally meaningful inter-β-strand restraints that confirm the interpretation of the electron densities, even if the assignment ambiguities did not allow for an NMR 3D structure determination.

Structure and distribution of amino acids in the new α-Syn fibril polymorphs.

Amino acids are colored in blue for positively charged, in red for negatively charged, in green for polar (including glycine), and in white for hydrophobic residues. Even and odd numberings are given on one monomer each. (A and C) Backbone structure. (B) and (D) Surface view.

Cryo-EM cross-sections of fibrils, formed by E46K, p-S129 phosphorylated, and N-terminally acetylated α-Syn protein.

Fibrils formed by E46K mutant α-Syn protein (A), Ser129 phosphorylated α-Syn protein (B), and N-terminally acetylated α-Syn protein (C) were analyzed by cryo-EM. Image processing did not allow reaching sufficient resolution for model building, but the cross-sections of the obtained 3D reconstructions are compatible with polymorph 2a for all three forms.

Figure 4 with 1 supplement
Schematic representation of α-Syn polymorphs.

(A) Diagram representing α-Syn regions with the N-terminus in blue, the NAC region in red and the C-terminus in yellow. (B) Representation of α-Syn fibril polymorphs 1a (PDB ID 6h6b; Guerrero-Ferreira et al., 2018), 1b (PDB ID 6cu8; Li et al., 2018a), 2a (PDB ID 6rt0, this work), and 2b (PDB ID 6rtb, this work), highlighting the striking differences in protofilament folding in α-Syn polymorphs 1a and 1b, compared to α-Syn polymorphs 2a and 2b. The atomic models obtained by cryo-EM of α-Syn polymorph 1a, polymorph 1b (Li et al., 2018a; Li et al., 2018b) and α-Syn polymorphs 2a and 2b (this work). Protein Data Bank (PDB) accession numbers are indicated.

Figure 4—figure supplement 1
Comparison between polymorphs 1 and 2.

Structures of polymorphs 1a, b and 2a, b with backbone shown in alternating colors in order to distinguish the location of the segments in the different structures. (A) Diagram representing α-Syn color coding from N-terminus to C-terminus: 1–10, yellow; 11–20, blue; 21–30, pink; 31–40, orange; 41–50 green; 51–60, black; 61–70, cyan; 71–80, light green; 81–90, red; 91–100 magenta; 101–140, gray. (B) Worm representation of α-Syn fibril polymorphs 1a, 1b, 2a and 2b. N-terminal and C-terminal residues number are indicated. Residues E53 and A53 are shown by van-der-Waals representation of the residues in green and blue respectively. Other residues are given in stick representation. Polymorph type, and PDB accession codes are indicated.

Videos

Video 1
Comparison of cryo-EM maps of α-Syn fibril polymorphs.

Cryo-EM reconstructions of α-Syn fibrils at 3.0 Å (polymorph 2a) and 3.4 Å (polymorph 2b) resolution detailing the interaction between two protofilaments (blue and orange) in each fibril, the 4.8 Å spacing between ß-strands and the topology of α-Syn monomers within a single protofilament.

Video 2
Structural differences between α-Syn polymorphs.

Atomic models of α-Syn fibrils represented as rounded ribbons with the N-terminus in blue and the NAC region in red.

Tables

Table 1
Growth conditions for α-Syn fibrils.
StudyBuffer compositionpHTemp–eratureTimeConcen–trationMethodα-Syn typePoly–morphPDB
This study50 mM Tris-HCl
150 mM KCl
7.537°C1 week (600 r.p.m.)700 µMCryo-EM + NMRFull-length (‘named fibrils’)2a, 2b6ssx
6sst
(Guerrero-Ferreira et al., 2018)DPBS (Gibco)
2.66 mM KCl,
1.47 mM KH2PO4,
137.93 mM NaCl, 8.06 mM Na2HPO4
7 to 7.337°C5 days (1000 r.p.m.)360 µM
(5 mg/mL)
Cryo-EMTruncated
(1-121)
1a6h6b
(Li et al., 2018a)50 mM Tris, 150 mM KCl,
0.05% NaN3
7.537°C3 days (900 r.p.m.)500 µMCryo-EMFull-length,
N-terminal acetylated
1a6a6b
(Li et al., 2018b)15 mM tetrabutyl–phosphonium bromideNot speci–fiedRoom tempe–rature14–30 days (quiescent)300 µMCryo-EMFull-length1a,b6cu7
6cu8
(Tuttle et al., 2016)50 mM sodium phosphate
0.12 mM EDTA
0.02% sodium azide (w/v)
7.437°C3 weeks (200 r.p.m.)1000 µM
(15 mg/mL)
NMRFull-length1a2n0a
(Rodriguez et al., 2015)5 mM lithium hydroxide
20 mM sodium phosphate
0.1 M NaCl
7.537°C72 hr500 µMMicro-EDPeptides: SubNACore, NACore, PreNAC4rik
4ril
4znn
(Rodriguez et al., 2015)50 mM Tris
150 mM KCl
7.537°C72 hr500 µMNo structureFull-length
(Gath et al., 2014a)50 mM Tris-HCl
150 mM KCl
7.537°C4 days (600 r.p.m.)300 µMNMR secondary structureFull-length2
(Gath et al., 2012)5 mM Tris-HCl7.537°C7 days (600 r.p.m.)300 µMNMR secondary structureFull-lengthDifferent from 1, 2
(Verasdonck et al., 2016)5 mM NaPO4937°C4 days (600 r.p.m.)300 µMNMR secondary structureFull-length1
This studyDPBS (Gibco):
2.66 mM KCl,
1.47 mM KH2PO4,
137.93 mM NaCl, 8.06 mM Na2HPO4
7 to 7.337°C5 days (1000 r.p.m.)360 µM
(5 mg/mL)
Cryo-EMFull-length, E46K2a
This studyDPBS (Gibco):
2.66 mM KCl,
1.47 mM KH2PO4,
137.93 mM NaCl, 8.06 mM Na2HPO4
7 to 7.337°C5 days (1000 r.p.m.)360 µM
(5 mg/mL)
Cryo-EMFull-length,
N-terminal acetylated
2a
 This studyDPBS (Gibco):
2.66 mM KCl,
1.47 mM KH2PO4,
137.93 mM NaCl, 8.06 mM Na2HPO4
7 to 7.337°C5 days (1000 r.p.m.)360 µM
(5 mg/mL)
Cryo-EMFull-length,
Phosphorylation at position S129
2a
Table 2
Cryo-EM structure determination statistics.
E46K mutantPhosphorylatedN-terminal acetylatedα-Syn polymorph 2aα-Syn polymorph 2b
Data Collection
Pixel size [Å]0.8310.8310.8310.6290.629
Defocus Range [µm]−0.8 to −2.5−0.8 to −2.5−0.8 to −2.5−0.8 to −2.5−0.8 to −2.5
Voltage [kV]300300300300300
Exposure time [s per frame]0.20.20.20.20.2
Number of frames5050505050
Total dose [e-2]6969696969
Reconstruction
Box width [pixels]280280280280280
Inter-box distance [pixels]2828282828
Micrographs8431’8879481’1431’143
Manually picked fibrils2’7025’0953’7515’2335’233
Initially extracted segments65’893107’14443’276100’323100’323
Segments after 2D classification50’514107’12635342100’193100’193
Segments after 3D classification50’51421’6853534219’9373’989
Resolution after 3D refinement [Å]4.654.654.753.343.75
Final resolution [Å]4.564.314.392.993.39
Estimated map sharpening B-factor [Å2]−208.6−104.3−178.1−67.1−76.4
Helical rise [Å]4.854.844.784.802.40
Helical twist [°]−0.79−0.77−0.71−0.80179.55
Table 3
Model building statistics.
α-Syn polymorph 2aα-Syn polymorph 2b
Initial model used [PDB code]6ssx6sst
Model resolution [Å]
(FSC = 0.143)
2.983.4
Model resolution range [Å]2.983.1
Map sharpening B-factor [Å2]−67.1−76.4
Model composition
Non-hydrogen atoms
Protein residues
Ligands
4900
730
0
4900
730
0
B-factors [Å2] (non-hydrogen atoms)
Protein
Ligand
48.18
-
70.32
-
R.m.s. deviations
Bond lengths [Å]
Bond angles [°]
0.008
0.938
0.009
0.941
Validation
MolProbity score
Clashscore
Poor rotamers [%]
2.43
6.14
4.26
2.11
5.73
2.13
Ramachandran plot
Favored [%]
Allowed [%]
Disallowed [%]
87.39
12.61
0.00
89.86
10.14
0.00
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (E. coli)BL21(DE3)StratageneAgilent Technology #200131Expression performed in LB medium
Transfected construct (pET-3a)pET3aNovagenhttps://www.addgene.org/vector-database/2637/Encoding full length human alpha synuclein asyn (UniProtKB - P37840) with a silent mutagenesis of codon 136 (TAC to TAT)
Transfected construct (pRT21)pRT21(Masuda et al., 2006)Full length human alpha synuclein asyn (UniProtKB - P37840) with a silent mutagenesis of codon 136 (TAC to TAT)
Transfected construct (pNatB)pNatB(Johnson et al., 2010)http://www.addgene.org/53613/Expression of the fission yeast NatB complex - chloramphenicol marker
Chemical compound, drugDEAE sepharose fast flowGE Healthcare, #17-0709-01
OtherCopper/carbon gridshttps://www.quantifoil.com/R 2/2 grids
Software, algorithmUCSF Chimera(Pettersen et al., 2004)https://www.cgl.ucsf.edu/chimeraRRID:SCR_004097
Software, algorithmSerialEM(Mastronarde, 2005)https://bio3d.colorado.edu/SerialEM/RRID:SCR_017293
Software, algorithmFOCUS(Biyani et al., 2017)http://focus-em.org
Software, algorithmMotionCor2(Zheng et al., 2017)https://emcore.ucsf.edu/ucsf-motioncor2RRID:SCR_016499
Software, algorithmRELION 2, 3(Scheres, 2012;
Zivanov et al., 2018)
http://www2.mrc-lmb.cam.ac.uk/relionRRID:SCR_016274
Software, algorithmCOOT(Emsley and Cowtan, 2004)https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/RRID:SCR_014222
Software, algorithmMolprobity(Williams et al., 2018)http://molprobity.biochem.duke.eduRRID:SCR_014226

Data availability

Raw cryo-EM micrographs are available in EMPIAR, entry numbers EMPIAR-10323. The 3D maps are available in the EMDB, entry numbers EMD-10307 (α-Syn polymorph 2a) and EMD-10305 (α-Syn-polymorph 2b). Atomic coordinates are available at the PDB with entry numbers PDB 6SSX (α-Syn polymorph 2a) and PDB 6SST (α-Syn polymorph 2b).

The following data sets were generated
  1. 1
  2. 2
  3. 3
  4. 4
  5. 5

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