Journal: Science / Year: 2020 Title: Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in . Authors: Ruben Hervas / Michael J Rau / Younshim Park / Wenjuan Zhang / Alexey G Murzin / James A J Fitzpatrick / Sjors H W Scheres / Kausik Si / Abstract: How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a ...How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo-electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and "seed" for further translationally active aggregation. The 31-amino acid protofilament core adopted a cross-β unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory.
Evidence: Cryo-Electron Microscopy Negative-Stain Electron Microscopy SDD-AGE PAGE SDS-PAGE Antidody reactivity ThT binding Proteinase K resistance
Type
Name
Symmetry operation
Number
identity operation
1_555
1
Buried area
20530 Å2
ΔGint
33 kcal/mol
Surface area
13880 Å2
Symmetry
Helical symmetry: (Circular symmetry: 3 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 9 / Rise per n subunits: 4.75 Å / Rotation per n subunits: -1.55 °)
-
Components
#1: Protein/peptide
Translationalregulatororb2
Mass: 3974.170 Da / Num. of mol.: 9 / Fragment: UNP residues 176-206 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly) / References: UniProt: Q9VSR3
-
Experimental details
-
Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction
-
Sample preparation
Component
Name: amyloid core of Drosophila Orb2 / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)
Organism: Drosophila melanogaster (fruit fly) / Tissue: head
Buffer solution
pH: 7.25
Specimen
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
Details: unspecified
Vitrification
Cryogen name: ETHANE
-
Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD
Image recording
Electron dose: 63 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19509 / Symmetry type: HELICAL
Refinement
Resolution: 2.6→86.72 Å / Cor.coef. Fo:Fc: 0.854 / WRfactor Rwork: 0.322 / SU B: 10.458 / SU ML: 0.21 / Average fsc overall: 0.8918 / Average fsc work: 0.8918 / ESU R: 0.37 Details: Hydrogens have been added in their riding positions
Rfactor
Num. reflection
% reflection
Rwork
0.3216
24927
-
all
0.322
-
-
Rfree
-
-
0 %
obs
-
-
100 %
Solvent computation
Solvent model: BABINET MODEL
Displacement parameters
Biso mean: 46.615 Å2
Baniso -1
Baniso -2
Baniso -3
1-
3.964 Å2
-0.057 Å2
-0.012 Å2
2-
-
3.926 Å2
-0.015 Å2
3-
-
-
-7.89 Å2
Refine LS restraints
Refine-ID
Type
Dev ideal
Dev ideal target
Number
ELECTRONMICROSCOPY
r_bond_refined_d
0.013
0.012
2865
ELECTRONMICROSCOPY
r_angle_refined_deg
0.998
1.616
3861
ELECTRONMICROSCOPY
r_dihedral_angle_1_deg
5.582
5
324
ELECTRONMICROSCOPY
r_dihedral_angle_2_deg
32.937
26.667
270
ELECTRONMICROSCOPY
r_dihedral_angle_3_deg
16.633
15
513
ELECTRONMICROSCOPY
r_chiral_restr
0.131
0.2
306
ELECTRONMICROSCOPY
r_gen_planes_refined
0.012
0.02
2484
ELECTRONMICROSCOPY
r_nbd_refined
0.153
0.2
1810
ELECTRONMICROSCOPY
r_nbtor_refined
0.268
0.2
3004
ELECTRONMICROSCOPY
r_xyhbond_nbd_refined
0.106
0.2
206
ELECTRONMICROSCOPY
r_mcbond_it
5.523
3.952
1188
ELECTRONMICROSCOPY
r_mcangle_it
8.553
5.836
1494
ELECTRONMICROSCOPY
r_scbond_it
8.576
4.898
1677
ELECTRONMICROSCOPY
r_scangle_it
13.629
7.077
2349
ELECTRONMICROSCOPY
r_lrange_it
17.636
53.966
3568
LS refinement shell
Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %
Resolution (Å)
Rfactor Rwork
Num. reflection Rwork
Rfactor all
Num. reflection all
Fsc work
WRfactor Rwork
2.6-2.667
0.612
1817
0.612
1817
0.717
0.612
2.667-2.741
0.593
1796
0.593
1796
0.726
0.593
2.741-2.82
0.439
1819
0.439
1819
0.843
0.439
2.82-2.907
0.409
1642
0.409
1642
0.873
0.409
2.907-3.002
0.401
1602
0.401
1602
0.858
0.401
3.002-3.107
0.375
1666
0.375
1666
0.918
0.375
3.107-3.224
0.278
1523
0.278
1523
0.934
0.278
3.224-3.356
0.252
1446
0.252
1446
0.939
0.252
3.356-3.505
0.262
1452
0.262
1452
0.944
0.262
3.505-3.675
0.248
1329
0.248
1329
0.958
0.248
3.675-3.874
0.235
1268
0.235
1268
0.96
0.235
3.874-4.108
0.223
1256
0.223
1256
0.96
0.223
4.108-4.391
0.23
1099
0.23
1099
0.956
0.23
4.391-4.742
0.279
1133
0.279
1133
0.969
0.279
4.742-5.193
0.246
934
0.246
934
0.95
0.246
5.193-5.803
0.208
903
0.208
903
0.97
0.208
5.803-6.696
0.414
784
0.414
784
0.893
0.414
6.696-8.189
0.404
645
0.404
645
0.866
0.404
8.189-11.53
0.501
519
0.501
519
0.889
0.501
11.53-86.72
0.773
294
0.773
294
0.934
0.773
+
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