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

Journal: Nucleic Acids Research

Article Title: Cryo-EM structures reveal a conserved architecture for raiA noncoding RNA

doi: 10.1093/nar/gkag185

Figure Lengend Snippet: Cryo-EM structure of raiA motif RNA from Nocardioides sp. Iso805N ( Ns-raiA ) at 3.0 Å resolution. ( A ) Cryo-EM density map of Ns-raiA shown in three different views. Individual stem–loops are color-coded as indicated. Pyramid diagrams indicate the orientation of Ns-raiA structure. ( B ) Overlaid cryo-EM densities and models of P1c, P3a, PK1, P5, P6, and P8.

Article Snippet: RNA samples were diluted to ∼30 μM in EM buffer (20 mM HEPES–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl 2 , 0.05% Igepal CA-630) before preparing cryo-EM samples.

Techniques: Cryo-EM Sample Prep

Journal: Nucleic Acids Research

Article Title: Cryo-EM structures reveal a conserved architecture for raiA noncoding RNA

doi: 10.1093/nar/gkag185

Figure Lengend Snippet: Tertiary and secondary structures of Ns-raiA . ( A ) Atomic model of Ns-raiA shown in three different views. Individual stem–loops are color-coded as indicated. Nucleotides in the conserved UU AGAC GUAA linker connecting PK1 and P6 not resolved in the cryo-EM map are shown as a dotted line. Secondary structures of Ns-raiA are shown in the canonical layout ( B ) as proposed in or in a layout that more closely reflects the tertiary structure ( C ). The nucleotides are colored as in the structure in panel (A). Black arrowheads indicate the backbone direction. Non-Watson–Crick base pairs are indicated with Leontis–Westhof nomenclature symbols (inset) .

Article Snippet: RNA samples were diluted to ∼30 μM in EM buffer (20 mM HEPES–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl 2 , 0.05% Igepal CA-630) before preparing cryo-EM samples.

Techniques: Cryo-EM Sample Prep

Journal: Nucleic Acids Research

Article Title: Cryo-EM structures reveal a conserved architecture for raiA noncoding RNA

doi: 10.1093/nar/gkag185

Figure Lengend Snippet: Structural comparison of raiA motif RNAs from Nocardioides sp. Iso805N ( Ns-raiA ) , Clostridium acetobutylicum ( Ca-raiA ), and Mogibacterium pumilum ( Mp-raiA ). Cryo-EM density map (left) and atomic model (right) of Ns-raiA ( A ), Ca-raiA ( B ), and Mp-raiA ( C ). The absence of P2 and distal P7 in Ca-raiA and Mp-raiA , and the absence of P8 in Mp-raiA are indicated by dashed lines. Sequence and secondary structure of Ca-raiA ( D ), and Mp-raiA ( E ). Black arrowheads indicate the backbone direction. Non-Watson–Crick base pairs are labeled as indicated. Insets show the schematics of J1 regions. Zoom-in views of the P1c-P2-P3a junction in Ns-raiA ( F ), and the P1c-P3a junctions in Ca-raiA ( G ) and Mp-raiA ( H ), highlighting the GAA(A) tetraloop fold, shown in the same orientation. Zoom-in views of the interface of P8 and PK1 stems in Ns-raiA ( I ) and Ca-raiA ( J ), and the PK1 stem in Mp-raiA ( K ), shown in the same orientation.

Article Snippet: RNA samples were diluted to ∼30 μM in EM buffer (20 mM HEPES–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl 2 , 0.05% Igepal CA-630) before preparing cryo-EM samples.

Techniques: Comparison, Cryo-EM Sample Prep, Sequencing, Labeling

Journal: Nucleic Acids Research

Article Title: Cryo-EM structures reveal a conserved architecture for raiA noncoding RNA

doi: 10.1093/nar/gkag185

Figure Lengend Snippet: Structural details of P1 and its interactions with the core. ( A ) Overall view of P1 (shown as colored ribbon for backbone and filled bases and sugars) and its position relative to the core (colored ribbon) in the structure of Ns-raiA . Other stems are shown as white ribbons. ( B ) Close-up view of the interface between P1 and the core, in dashed box region in panel (A). The two insert panels highlight the long-range A81-G240-G10 stacking and the G10-G14-C242 base triple, respectively. ( C ) Secondary structure representation of the region shown in panel (B). Long-range stacking interactions are indicated by gray dashed lines, while base triple interactions are marked with green lines. ( D ) Representative 2D class averages of To-raiA (left) and enlargement with structure features labeled (right). ( E ) Cryo-EM density map and ribbon model of To-raiA . ( F ) Sequence conservation of raiA motif RNA mapped onto the Ns-raiA structure. View on left highlights conservation of P1 and on right conservation of the core. Conservation scores were calculated using the ConSurf server .

Article Snippet: RNA samples were diluted to ∼30 μM in EM buffer (20 mM HEPES–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl 2 , 0.05% Igepal CA-630) before preparing cryo-EM samples.

Techniques: Labeling, Cryo-EM Sample Prep, Sequencing