Journal: Cell

Article Title: BacPROTACs mediate targeted protein degradation in bacteria

doi: 10.1016/j.cell.2022.05.009

Figure Lengend Snippet: BacPROTAC-1 induces formation of an active ClpC oligomer, related to Figure 2 (A) Negative-stained EM analysis of B. subtilis ClpC DWB in the absence of BacPROTAC-1: a representative micrograph from the 1,004 collected (scale bar, 350 Å). (B) Cryo-EM analysis of B. subtilis ClpC DWB in the presence of BacPROTAC-1 and mSA-Kre: a representative micrograph from the 4,455 collected (scale bar, 420 Å). (C) Negative-stain EM analysis of B. subtilis ClpC DWB :ClpP in the presence of BacPROTAC-1: a representative micrograph of the 84 micrographs collected (scale bar, 850 Å). (D) SEC analysis of ClpC DWB in the presence of BacPROTAC-1 (cyan) compared with the SEC analysis reported in Figure 2 A of a stoichiometric ClpC DWB :mSA-Kre mixture in the presence (gray) or absence (gray, dotted line) of BacPROTAC-1. The fraction used for negative-stained EM analysis is highlighted. A representative micrograph area and SDS-PAGE gel are shown in (E), together with representative 2D class averages. (F) SEC analysis of ClpC DWB in the presence of equimolar amounts of pArg-β-casein (orange) compared with the SEC analysis reported in Figure 2 A. The fraction used for negative-stain EM analysis is highlighted. A representative micrograph area and SDS-PAGE gel are shown in (G), together with representative 2D class averages.

Article Snippet: The quality of the grids was screened using a Thermo Scientific Glacios Cryo Transmission Electron Microscope equipped with a Falcon3 Direct Electron Detector.

Techniques: Staining, Cryo-EM Sample Prep, SDS Page

Journal: Cell

Article Title: BacPROTACs mediate targeted protein degradation in bacteria

doi: 10.1016/j.cell.2022.05.009

Figure Lengend Snippet: Cryo-EM data collection, refinement, and validation statistics

Article Snippet: The quality of the grids was screened using a Thermo Scientific Glacios Cryo Transmission Electron Microscope equipped with a Falcon3 Direct Electron Detector.

Techniques: Biomarker Discovery

Journal: Cell

Article Title: BacPROTACs mediate targeted protein degradation in bacteria

doi: 10.1016/j.cell.2022.05.009

Figure Lengend Snippet: Cryo-EM structure of the activated ClpC hexamer in complex with a BacPROTAC-tethered substrate (A) Representative 2D class averages from cryo-EM images are shown together with the final 3D map at a resolution of 3.7 Å. The density is colored according to subunits that are termed P1–P6. The substrate captured in the central channel is shown in yellow. (B) Side views of the substrate-bound ClpC. The lower one shows the cross section of the hexamer highlighting the substrate threaded through the two D1 and D2 rings of ClpC. The substrate was well defined by cryo-EM density over the entire passage of the central channel (80 Å). (C) Arrangement of primary D1 and D2 pore loops engaging the substrate (peptide backbone shown in yellow). The P6 pore loops, which were not in contact with the substrate, were too flexible to be modeled into the cryo-EM density. Their approximate position is indicated by flanking residues. (D) Cryo-EM density of the tyrosine-bearing pore loops overlaid with the final model. (E) Schematic representation of nucleotide states and substrate engagement of the six ClpC protomers. Nucleotides were assigned based on cryo-EM density and distance matrices in the active site ( Figure S3 D). See also Figure S3 .

Article Snippet: The quality of the grids was screened using a Thermo Scientific Glacios Cryo Transmission Electron Microscope equipped with a Falcon3 Direct Electron Detector.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: BacPROTACs mediate targeted protein degradation in bacteria

doi: 10.1016/j.cell.2022.05.009

Figure Lengend Snippet: Cryo-EM structure of the activated ClpC hexamer in complex with a BacPROTAC-tethered substrate, related to Figure 3 (A) FSC curves of the final maps obtained by cryo-EM analysis, showing a resolution of 10 Å for the tetramer of hexamers map and 3.7 Å for the single hexamer map. (B) Angular distribution of the particles used to reconstruct the single hexamer map. (C) Local resolution map for the single hexamer in different orientations. (D) Nucleotide-binding sites of the six ClpC DWB protomers. The different panels show the modeled nucleotide in each active site pocket and some of the crucial residues involved in ATP hydrolysis and ATP/ADP interaction. Contacts between ATP γ-phosphate and Arg fingers in D1 (R332-R333) and D2 (R704) are indicated ( ∗ ) for the ATP-bound sites, while distances between Arg fingers Cα and ADP β-phosphate are shown for ADP-bound sites. The cryo-EM map is represented as gray surface around the modeled protein structure.

Article Snippet: The quality of the grids was screened using a Thermo Scientific Glacios Cryo Transmission Electron Microscope equipped with a Falcon3 Direct Electron Detector.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Cell

Article Title: BacPROTACs mediate targeted protein degradation in bacteria

doi: 10.1016/j.cell.2022.05.009

Figure Lengend Snippet:

Article Snippet: The quality of the grids was screened using a Thermo Scientific Glacios Cryo Transmission Electron Microscope equipped with a Falcon3 Direct Electron Detector.

Techniques: Virus, Recombinant, Electron Microscopy, Plasmid Preparation, Software, Variant Assay