mtb cryo em buffer  (Thermo Fisher)

Journal: bioRxiv

Article Title: RNA polymerase inhibitors reveal active-site motions essential for the nucleotide-addition cycle

doi: 10.64898/2026.04.06.716786

Figure Lengend Snippet:

Article Snippet: Mtb RNAP was dialyzed overnight into Mtb cryo-EM buffer (20 mM HEPES, pH 7.5, 150 mM potassium glutamate, 5 mM magnesium acetate, 2.5 mM DTT). t-DNA:RNA hybrid was added to the RNAP at a 1.1x molar excess to RNAP and incubated at RT for 15 min. nt-DNA was added in 1.5x molar excess to t-DNA and incubated at RT for 15 min.

Techniques: Biomarker Discovery

Journal: bioRxiv

Article Title: RNA polymerase inhibitors reveal active-site motions essential for the nucleotide-addition cycle

doi: 10.64898/2026.04.06.716786

Figure Lengend Snippet: (A-C) Overall cryo-EM structures and active-site conformations of Eco -ePECs. Left: The cryo-EM maps are colored by RNAP subunit or key structural motifs. α and ꞷ are light gray, β light blue, β’ light pink, RH is crimson, FL orange, TL magenta, and SI3 is tan. Right: Close-up and rotated view of active site regions shown using secondary structure cartoons. Nucleic acid color-coding: RNA red, t-DNA black. The RNAP active-site Mg 2+ is a yellow sphere. Arrows indicate open or closed RH-FL, TL and SI3. (A) Open active-site conformation (RH-FL open, TL open, SI3 open). (B) Closed active-site conformation (RH-FL closed, TL closed, SI3 closed). (C) SemiClosed active-site conformation (RH-FL open, TL closed, SI3 closed). (D) Overall cryo-EM structure Open Eco -ePEC(CBR9379). (E) Close-up and rotated view of CBR9379-bound region of Eco RNAP. Side chains of residues that interact with CBR9379 (green) are shown. Map density for CBR9379 is shown. (F) Pie charts illustrating cryo-EM particle distributions for Eco -ePEC into Open, Closed and SemiClosed active-site states -/+ CBR9379.

Article Snippet: Mtb RNAP was dialyzed overnight into Mtb cryo-EM buffer (20 mM HEPES, pH 7.5, 150 mM potassium glutamate, 5 mM magnesium acetate, 2.5 mM DTT). t-DNA:RNA hybrid was added to the RNAP at a 1.1x molar excess to RNAP and incubated at RT for 15 min. nt-DNA was added in 1.5x molar excess to t-DNA and incubated at RT for 15 min.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: RNA polymerase inhibitors reveal active-site motions essential for the nucleotide-addition cycle

doi: 10.64898/2026.04.06.716786

Figure Lengend Snippet: (A) Elongation scaffold with 3’-deoxy RNA (3’-dG20-RNA) used for cryo-EM of Mtb RNAP. nt-DNA is gray, t-DNA is black, RNA is red. Incoming GTP added during grid preparation is indicated. (B-D) Overall cryo-EM structures and active-site conformations of Mtb -ECs. Left: The cryo-EM maps are colored by RNAP subunit or key structural motifs. α and ꞷ are light gray, β light blue, β’ light pink, RH is crimson, FL orange, TL magenta. Right: Close-up and rotated view of active site regions shown using secondary structure cartoons. Nucleic acid color-coding: RNA red, t-DNA black. The RNAP active-site Mg 2+ is a yellow sphere. Arrows indicate open or closed RH-FL, TL and SI3. (B) Open active-site conformation (RH-FL open, TL open). (C) Closed active-site conformation (RH-FL closed, TL closed). (D) SemiClosed active-site conformation (RH-FL open, TL closed). (E) Overall cryo-EM structure Open Mtb -EC(AAP-SO 2 ). (F) Close-up and rotated view of AAP-SO 2 -bound region of Mtb RNAP. Side chains of residues that interact with AAP-SO 2 (green) are shown. Map density for AAP-SO 2 is shown. (G) Pie charts illustrating cryo-EM particle distributions for Mtb -EC into Open, Closed and SemiClosed active-site states -/+ AAP-SO 2 .

Article Snippet: Mtb RNAP was dialyzed overnight into Mtb cryo-EM buffer (20 mM HEPES, pH 7.5, 150 mM potassium glutamate, 5 mM magnesium acetate, 2.5 mM DTT). t-DNA:RNA hybrid was added to the RNAP at a 1.1x molar excess to RNAP and incubated at RT for 15 min. nt-DNA was added in 1.5x molar excess to t-DNA and incubated at RT for 15 min.

Techniques: Cryo-EM Sample Prep