pshs325  (Addgene inc)

Journal: Nature Communications

Article Title: Backbone-independent NMR resonance assignments of methyl probes in large proteins

doi: 10.1038/s41467-021-20984-0

Figure Lengend Snippet: Performance comparison of different methyl assignment programs.

Article Snippet: The coding sequences for Cas9 HNH (residues 776–908) and REC2 domains (residues 167–307) were synthesized, codon optimized for expression in E. coli , and subcloned into a His 10 -MBP expression vector (Genscript) (Supplementary Table ). pSHS325 bacterial expression plasmid for SpCas9 REC3 domain was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid #101205) .

Techniques: Comparison, Labeling

Journal: Nature Communications

Article Title: Backbone-independent NMR resonance assignments of methyl probes in large proteins

doi: 10.1038/s41467-021-20984-0

Figure Lengend Snippet: MAUS methyl resonance assignment statistics.

Article Snippet: The coding sequences for Cas9 HNH (residues 776–908) and REC2 domains (residues 167–307) were synthesized, codon optimized for expression in E. coli , and subcloned into a His 10 -MBP expression vector (Genscript) (Supplementary Table ). pSHS325 bacterial expression plasmid for SpCas9 REC3 domain was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid #101205) .

Techniques: Labeling

Journal: Nature Communications

Article Title: Backbone-independent NMR resonance assignments of methyl probes in large proteins

doi: 10.1038/s41467-021-20984-0

Figure Lengend Snippet: a Domain organization of Spy Cas9 composed of the recognition lobe (REC) and nuclease lobe (NUC). BH, bridge helix; PI, PAM-interacting. b Surface representation of SpyCas9 (PDB ID 4cmp) depicting the bilobed architecture. Protein domains are colored as in a . c Single domains used in the divide-and-conquer approach. 1 H- 13 C HMQC spectra of selectively labeled HNH (green), REC2 (red), and REC3 (blue) domains at Ile δ 1 - 13 CH 3 ; Leu, Val- 13 CH 3 / 13 CH 3 positions, acquired at 800 MHz, 25 °C. The spectra of the individual domains of Cas9 indicate that they retain their proper fold when in isolation. d Number of valid assignment options for each residue identified by MAUS for HNH, REC2, and REC3, respectively. The colored spheres represent final valid resonance assignment options: violet (1 option), green (2 options), yellow (3 options), and red (>3 options).

Article Snippet: The coding sequences for Cas9 HNH (residues 776–908) and REC2 domains (residues 167–307) were synthesized, codon optimized for expression in E. coli , and subcloned into a His 10 -MBP expression vector (Genscript) (Supplementary Table ). pSHS325 bacterial expression plasmid for SpCas9 REC3 domain was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid #101205) .

Techniques: Labeling, Isolation, Residue