3d em reconstructions  (Broad Institute Inc)

Journal: bioRxiv

Article Title: Structural architecture of amyloid-β oligomers, curvilinear protofibrils and annular assemblies, imaged by cryo-EM and cryo-ET

doi: 10.1101/2024.03.01.582902

Figure Lengend Snippet: Overview of prefibrillar assemblies as a heterogeneous snap-shot, imaged by cryo-EM. Typical classification of assemblies, and approximate number of Aβ 42 monomers is indicated (A). Abinitio 3D reconstruction from 2.5 million particles, 3 structures from 10 are shown. Oligomer (blue) from 180k particles; Curvilinear Protofibril (green) 322k particles and Annular assembly (pink) 298 particles. Scale Bar 20 Å for 2D class averages; 50 Å for 3D structures.

Article Snippet: 7 nm, which closely matches the cryo-EM 3D reconstructions shown in and S5, the length of the internal channel appears a little smaller at ca .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural architecture of amyloid-β oligomers, curvilinear protofibrils and annular assemblies, imaged by cryo-EM and cryo-ET

doi: 10.1101/2024.03.01.582902

Figure Lengend Snippet: Cryo-EM 2D class averages of typical oligomers and curvilinear protofibrils. 209,000 particles in total, scale bar 20 Å. The class averages have been arranged in increasing length. Top row 25-50 Å, second row 60-80 Å; 3 rd row 90-105 Å; and bottom row 100-140 Å, (A). Distribution of oligomer and curvilinear protofibrils length. Taken from half a million particles; 95 % of particles are less than 90 Å long (B). Density profiles of the protofibril diameter. Six profiles for each row (Red, orange yellow left side images. Green, blue and purple on the right side) (C). Diameters observed for each row. The mean diameter for class averages in rows 2, 3 and 4 are consistently 28 +/-1 Å (standard dev.) (D).

Article Snippet: 7 nm, which closely matches the cryo-EM 3D reconstructions shown in and S5, the length of the internal channel appears a little smaller at ca .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural architecture of amyloid-β oligomers, curvilinear protofibrils and annular assemblies, imaged by cryo-EM and cryo-ET

doi: 10.1101/2024.03.01.582902

Figure Lengend Snippet: Cryo-ET 3D tomograms. (A) Typical oligomers, side and orthogonal top view are shown (top row), and curvilinear protofibrils (row 2 & 3). The cryo-ET single particles have a close resemblance to the cryo-EM class averages, shown in . Lengths of the long axis are: Top row 25-50 Å; 2 nd row 60-100 Å; 3 rd row 100-300 Å. Images are presented as sums of 5-10 2D slices, each 7.8 Å thick. Scale Bar = 100 Å. (B) Density profiles show that the particles have a consistent diameter of ca . 2.8 nm.

Article Snippet: 7 nm, which closely matches the cryo-EM 3D reconstructions shown in and S5, the length of the internal channel appears a little smaller at ca .

Techniques: Tomography, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural architecture of amyloid-β oligomers, curvilinear protofibrils and annular assemblies, imaged by cryo-EM and cryo-ET

doi: 10.1101/2024.03.01.582902

Figure Lengend Snippet: Cryo-EM 3D reconstruction of Aβ 42 annular oligomers. 2D averages (A) and 3D reconstruction (B) from 6k single particles suggests the structure has an internal pore. External Diameter, of top view is 65-55 Å; Length of channel is 54 Å; Volume suggests 12-16 Aβ 42 molecules. Scale bar: 50 Å for 3D structures, 20 Å for 2D averages.

Article Snippet: 7 nm, which closely matches the cryo-EM 3D reconstructions shown in and S5, the length of the internal channel appears a little smaller at ca .

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Posttranslational modifications mediate the structural diversity of tauopathy strains

doi: 10.1016/j.cell.2020.01.027

Figure Lengend Snippet: (A-D) Representative neuropathological changes in the frontal cortex of a patient with CBD. Ballooned neurons revealed by H&E staining (A, arrow) and immunolabeling with CP13 (B, arrow). CP13 immunostaining also labels neuritic threads (B-D), as well as pleomorphic small neuronal inclusions (C, arrows) and astrocytic plaques (D, asterisk). Scale bar equal to 20 microns (A-D). (E) Sarkosyl-insoluble material from the frontal cortex of the CBD patient used for cryo-EM analysis was evaluated by Western blot, including the tau antibodies PHF1 (pS396/404), Tau46 (aa404–441) and 12E8 (pS262, pS356). (F) Representative immuno-electron microscopy images of sarkosyl-insoluble tau filaments purified from CBD brain and stained with tau phospho-specific antibodies PHF1 and 12E8, as well as ubiquitin (Ubi-1). Primary antibodies were visualized with secondary antibody conjugated with 6 nm gold particles. Scale bar equal to 30 nm. (G) Representative cryo-EM images of the singlet and doublet tau fibrils in CBD. Scale bar equal to 100 Å. (H) Pronase treatment eliminates ubiquitin immunoreactivity from sarkosyl-insoluble core. To examine the impact of pronase treatment on ubiquitination of the filament core in tauopathies, the sarkosyl insoluble fraction was obtained and incubated in the presence or absence of pronase (1, 2, or 5 minute treatment), and subsequently evaluated by dot blot (including anti-ubiquitin and the tau antibodies E1 [aa19–33], CP13 [pS202], 12E8, PHF1, and Tau46). (I) Schematic diagram depicting the location of the antibody epitope within the tau protein. Note the amount of each sarkosyl-insoluble fraction was normalized for total tau levels. See also Figures S1 and S5.

Article Snippet: An average of 10 z-slices from the (A) CBD doublet fibril and (C) AD straight filament (EMD-3743) cryo-EM 3D reconstructions reveals strong, large densities visibly attached to K 321 and K 353 on the CBD doublet map and K 317 , K 321 , and K 311 on the AD straight filament (red dashed circles).

Techniques: Staining, Immunolabeling, Immunostaining, Cryo-EM Sample Prep, Western Blot, Immuno-Electron Microscopy, Purification, Incubation, Dot Blot

Journal: Cell

Article Title: Posttranslational modifications mediate the structural diversity of tauopathy strains

doi: 10.1016/j.cell.2020.01.027

Figure Lengend Snippet: Cryo-EM density (mesh) and atomic models (colored sticks) of (A) singlet and (B) doublet fibrils in CBD. Extra densities (pink mesh), which are directly connected, or in close proximity, to many sidechains in the cryo-EM maps, correspond to non-tau protein components. (C) Schematic view of the CBD doublet fibril. The extra, unknown densities are overlaid as capped pink mesh. See also Figure S2.

Article Snippet: An average of 10 z-slices from the (A) CBD doublet fibril and (C) AD straight filament (EMD-3743) cryo-EM 3D reconstructions reveals strong, large densities visibly attached to K 321 and K 353 on the CBD doublet map and K 317 , K 321 , and K 311 on the AD straight filament (red dashed circles).

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Posttranslational modifications mediate the structural diversity of tauopathy strains

doi: 10.1016/j.cell.2020.01.027

Figure Lengend Snippet: (A) Sequence alignment of the four microtubule-binding repeats (R1-R4) of tau and the sequence after R4 that is part of the CBD and AD fibril cores. The positions of filamentous β-strands in both diseases are shown. PTMs detected by MS in tau fibrils from CBD case 1 and AD, the structures of which were determined by cryo-EM in this work, are shown with acetylation, ubiquitination, trimethylation, and phosphorylation sites marked with blue, orange, red, and green balls, respectively. Sidechains with multiple PTMs detected are shown with two colours. Acetylation of K321 and K370 (Park, et al., 2018) and ubiquitination of K353 (Cripps et al., 2006) in the AD fibril core are included from the literature. PTMs are mapped onto schematics of the protofilament structures from (B) CBD case 1, and (C) AD. The same color scheme as described above is used to depict PTMs. See also Figures S4 and S7 and Tables S1, S2, S3, and S4.

Article Snippet: An average of 10 z-slices from the (A) CBD doublet fibril and (C) AD straight filament (EMD-3743) cryo-EM 3D reconstructions reveals strong, large densities visibly attached to K 321 and K 353 on the CBD doublet map and K 317 , K 321 , and K 311 on the AD straight filament (red dashed circles).

Techniques: Sequencing, Binding Assay, Cryo-EM Sample Prep

Journal: Cell

Article Title: Posttranslational modifications mediate the structural diversity of tauopathy strains

doi: 10.1016/j.cell.2020.01.027

Figure Lengend Snippet: An average of 10 z-slices from the (A) CBD doublet fibril and (C) AD straight filament (EMD-3743) cryo-EM 3D reconstructions reveals strong, large densities visibly attached to K321 and K353 on the CBD doublet map and K317, K321, and K311 on the AD straight filament (red dashed circles). In particular, note the large, near-stoichiometric densities at the interface between protofilaments (A, C). Many of these sites are detected to be ubiquitinated by MS and the lysine-connected densities are much too large to be an acetyl group. A large, buried density proximal to K290, K294, H362 and K370 in the CBD doublet fibril and a large, solvent-exposed density next to K353, H362 and K369 in the AD straight filament are also shown (green dashed circle). Schematics shown to scale in (B) and (D) highlight the structural role mono- or poly-ubiquitinated chains at these lysines may play in the CBD doublet fibril and AD straight filament, respectively. Brackets surrounding ubiquitin indicate the possibility of a (poly)-ubiquitin chain. The position of the K353 and H362 sidechains are shown as filled blue circles. Scale bar is equal to 25 Å. See also Figure S6.

Article Snippet: An average of 10 z-slices from the (A) CBD doublet fibril and (C) AD straight filament (EMD-3743) cryo-EM 3D reconstructions reveals strong, large densities visibly attached to K 321 and K 353 on the CBD doublet map and K 317 , K 321 , and K 311 on the AD straight filament (red dashed circles).

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Posttranslational modifications mediate the structural diversity of tauopathy strains

doi: 10.1016/j.cell.2020.01.027

Figure Lengend Snippet: An average of 10 z-slices from the (A) CBD singlet fibril and (C) AD tau fibril cryo-EM 3D reconstructions reveals strong, large densities visibly attached to K321, K343, K353, and K369 on the CBD singlet map and K317 and K321 on the AD tau protofilament (red dashed circles). Many of these sites are detected to be ubiquitinated by MS, and the lysine-connected densities are much too large to be an acetyl group. A large, buried density proximal to K290, K294, H362, and K370 in the CBD singlet fibril is also shown (green dashed circle). Schematics shown to scale in (B) and (D) highlight the structural role mono- or poly-ubiquitinated chains at these lysines may play in the CBD singlet fibril and AD tau protofilament, respectively. Brackets surrounding ubiquitin indicate the possibility of a (poly)-ubiquitin chain. The position of the H362 sidechain is shown as a filled blue circle. Scale bar is equal to 25 Å.

Article Snippet: An average of 10 z-slices from the (A) CBD doublet fibril and (C) AD straight filament (EMD-3743) cryo-EM 3D reconstructions reveals strong, large densities visibly attached to K 321 and K 353 on the CBD doublet map and K 317 , K 321 , and K 311 on the AD straight filament (red dashed circles).

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Posttranslational modifications mediate the structural diversity of tauopathy strains

doi: 10.1016/j.cell.2020.01.027

Figure Lengend Snippet: Based on our cryo-EM maps and MS PTM mapping onto atomic models, we conclude that ubiquitination of tau can mediate inter-protofilament interfaces in the doublet CBD fibril and straight filament from AD. If sites on tau favoring the formation of doublet fibrils in CBD (K353) or straight filaments in AD (K311 and K317/321) are acetylated, or ubiquitinated with low occupancy, this inter-protofilament interface is less likely to form. The singlet fibril in CBD does not bind to a second protofilament and paired helical filaments in AD have structures that do not require mediation by non-tau components at their inter-protofilament interface. The outcome of this model is that the incorporation of ubiquitin into tau filaments in CBD and AD mediates inter-protofilament packing resulting in distinct ultrastructural polymorphs, tuning the ratio of fibril subtypes in tau inclusions.

Article Snippet: An average of 10 z-slices from the (A) CBD doublet fibril and (C) AD straight filament (EMD-3743) cryo-EM 3D reconstructions reveals strong, large densities visibly attached to K 321 and K 353 on the CBD doublet map and K 317 , K 321 , and K 311 on the AD straight filament (red dashed circles).

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structure basis for directional R-loop formation and substrate handover mechanisms in Type I CRISPR-Cas system

doi: 10.1016/j.cell.2017.06.012

Figure Lengend Snippet: A. crRNA and dsDNA sequences used to program TfuCascade for structural studies. Residue numbers and color schemes are followed throughout the text. PAM and disordered NTS region are highlighted in yellow and grey boxes, respectively. B. The cryo-EM map of the TfuCascade/R-loop complex filtered to 3.3 Å without applying a B-factor, allowing the tracing of the entire R-loop. C. A representative region in the cryo-EM map of the TfuCascade/R-loop complex. D. Surface (left) and cartoon (right) representations revealing the subunit organization in TfuCascade, the elongated R-loop inside, and the orientation between PAM proximal and PAM distal dsDNA. E. Location of the PAM-distal dsDNA. F. Positively charged patches near the gapped NT region (NT strand binding site), the dsDNA entry site (K-vise), and above the target strand-binding groove (K-rim). G. Disrupting TfuCse2-specific structure features reduced R-loop formation efficiency. H. Alignment of TfuCascade/R-loop and Cas9/R-loop (PDB: 5F9R) structures along the PAM-proximal dsDNA revealing differences in the R-loop.

Article Snippet: The cryo-EM 3D reconstruction of this complex was generated at an overall resolution of 3.8 Å ( Figure S5 ), which allowed the unambiguous tracing of backbones and most side chains.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Cell

Article Title: Structure basis for directional R-loop formation and substrate handover mechanisms in Type I CRISPR-Cas system

doi: 10.1016/j.cell.2017.06.012

Figure Lengend Snippet: A. B. Two different views of the TfuCascade/seed-bubble cryo-EM structure highlighting the bending at the PAM region, extent of the seed-bubble, and the paths of the target and non-target strands. Type 2 class is illustrated here to emphasize the path of the non-target strand. C. Function of Q-wedge, L3-loop and Cas7.4 K-vise in shaping the path of the seed-bubble. D. Inclusion of a post-seed-bubble dsDNA to model the entire DNA substrate in the TfuCascade/seed-bubble complex. DNA bending around the PAM region likely plays an important role in melting dsDNA into a seed-bubble. E. DNA substrate follows a distinct path in the seed-bubble and full R-loop states. Note the differences in directionality, curvature, and DNA bending angle. F. Alanine substitution in K-vise reduced the target DNA binding affinity of TfuCascade by ~5-fold.

Article Snippet: The cryo-EM 3D reconstruction of this complex was generated at an overall resolution of 3.8 Å ( Figure S5 ), which allowed the unambiguous tracing of backbones and most side chains.

Techniques: Cryo-EM Sample Prep, Binding Assay