cryo em dataset  (Thermo Fisher)

Journal: Nature Communications

Article Title: Molecular basis of bacterial DSR2 anti-phage defense and viral immune evasion

doi: 10.1038/s41467-024-48291-4

Figure Lengend Snippet: A Domain organization of DSR2 (top) and the atomic model of DSR2 (bottom). The SIR2 domain, MD domain, and CTD domain are colored in salmon, light blue, and sky blue, respectively. B Top and side views of the cryo-EM density map of the apo DSR2 tetramer in the inactive state. Four DSR2s are colored in pale green, forest, light blue, and teal, respectively. The apo DSR2 tetramer has a bone-like supramolecular structure. C The atomic model of the apo DSR2 tetramer (left) and the SeThsA tetramer (right) (PDB ID 7UXT ). Four SIR2 domains form the tetrameric core in both the apo DSR2 tetramer and SeThsA (orange dashed boxes). The SLOG dimer of SeThsA is highlighted by a blue dashed circle. D Structural alignment of the SIR2 domains of apo SeThsA (light orange) and DSR2 (teal). Two types of SIR2 domains have similar structures and conserved active sites. The catalytic residues are shown as spheres, light pink for N113/H353 in SeThsA and magenta for N133/H171 in DSR2.

Article Snippet: Data collection of the cryo-EM datasets of DSR2 and DSR2-DSAD1 were performed on a 300 kV Titan Krios electron microscope (FEI) equipped with K3 and K2 Summit camera (Gatan) respectively, and a GIF Quantum energy filter operated with a slit width of 20 eV.

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Molecular basis of bacterial DSR2 anti-phage defense and viral immune evasion

doi: 10.1038/s41467-024-48291-4

Figure Lengend Snippet: A In vitro NAD + degradation assays of DSR2 with phage Tube protein. Apo DSR2 has no detectable NAD + cleavage activity. Neither the hexamer nor the filament of the Tube protein can activate the NAD + activity of apo DSR2. DSR2 + DSR2 H171A -Tube complex shows NAD + activity. DSR2 H171A -Tube complex abolishes NAD + cleavage activity. Wild-type DSR2 was used as control. Data are presented as means ± SD ( n = 5 independent experiments). B Representative 2D class average of Tube hexamer and Tube filament. C Schematic diagram of DSR2 recognizing the monomeric Tube but not the hexameric Tube or filament, to trigger NAD + hydrolysis. Top and side views of the cryo-EM density map ( D ) and atomic model ( E ) of DSR2 H171A -Tube complex. Four DSR2s are colored in pale green, forest, light blue, and teal; Tube proteins are colored in yellow.

Article Snippet: Data collection of the cryo-EM datasets of DSR2 and DSR2-DSAD1 were performed on a 300 kV Titan Krios electron microscope (FEI) equipped with K3 and K2 Summit camera (Gatan) respectively, and a GIF Quantum energy filter operated with a slit width of 20 eV.

Techniques: In Vitro, Activity Assay, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Molecular basis of bacterial DSR2 anti-phage defense and viral immune evasion

doi: 10.1038/s41467-024-48291-4

Figure Lengend Snippet: Top and side views of the cryo-EM density map ( A ) and the atomic model ( B ) of the DSR2-DSAD1 complex. Four DSR2s are colored in pale green, forest, light blue, and teal; DSAD1 proteins are colored in purple. C Schematic model of DSR2-DSAD1 co-complex formation resulting in inhibition of NAD + hydrolysis by DSR2 due to absence of Tube protein. Same color code as in ( A ); Tube is shown in yellow. D Structural alignment of a DSAD1 (purple) and Tube (yellow) both binding to the same site on DSR2 (teal). The steric clash between DSAD1 and Tube protein occurs at the same binding interface of the DSR2 CTD. E Overall structure of the DSR2-DSAD1 complex, DSR2s are colored in blue and cyan; DSAD1 proteins are colored in orange. F − H Detailed insights into the interactions between DSR2 and DSAD1 protein. Two major regions of hydrophobic interactions and one major region of hydrogen bonding interactions between DSR2 and DSAD1. Key interacting residues are shown in stick representation. Hydrogen bonds are shown in black dashed lines. I In vitro NAD + degradation assays of wild-type DSR2 and the DSR2-DSAD1 complex. DSR2-DSAD1 significantly reduces NAD + cleavage activity. Data are presented as means ± SE ( n = 5 independent experiments).

Article Snippet: Data collection of the cryo-EM datasets of DSR2 and DSR2-DSAD1 were performed on a 300 kV Titan Krios electron microscope (FEI) equipped with K3 and K2 Summit camera (Gatan) respectively, and a GIF Quantum energy filter operated with a slit width of 20 eV.

Techniques: Cryo-EM Sample Prep, Inhibition, Binding Assay, In Vitro, Activity Assay

Journal: Nature Communications

Article Title: Molecular basis of bacterial DSR2 anti-phage defense and viral immune evasion

doi: 10.1038/s41467-024-48291-4

Figure Lengend Snippet: A Top and side views of cryo-EM density maps of the apo DSR2 tetramer in the inactive state (top), the DSR2-DSAD1 complex in the inhibited state (middle), and the DSR2 H171A -Tube in the active state (bottom). Four DSR2s are colored in pale green, forest, light blue and teal, respectively. DSAD1 is colored in purple. Tube is colored in yellow. The conformation of the active state of DSR2 H171A -Tube was significantly more compact than the inactive and inhibited states. B Left, structural alignment of the protomers of apo DSR2 (light blue) and the active DSR2-Tube complex (brown for active DSR2 and yellow for Tube). Upon binding to the Tube protein, CTD circular solenoid lid of active DSR2 tilts ~30° to bind tightly to the Tube protein, while the SIR2 domain tilts ~13°. Right, structural alignment of the protomers of apo DSR2 (light blue) and DSR2-DSAD1 complex (purple for inhibited DSR2 and violet for DSAD1). C The interface between SIR2(a) and MD(b) from the adjacent DSR2(b). D The DSR2 loop143-148 , DSR2 I463G/Y471G , DSR2 N521G/F522G/M531G/P532G and DSR2 linker mutations at the SIR2(a)-MD(b) interface resulted in alterations in NAD + cleavage. Wild-type DSR2 was used as control. Data are presented as means ± SD ( n = 5 independent experiments).

Article Snippet: Data collection of the cryo-EM datasets of DSR2 and DSR2-DSAD1 were performed on a 300 kV Titan Krios electron microscope (FEI) equipped with K3 and K2 Summit camera (Gatan) respectively, and a GIF Quantum energy filter operated with a slit width of 20 eV.

Techniques: Cryo-EM Sample Prep, Binding Assay