Structural Determination of CRISPR HNH Domain

Following TEV cleavage of the His₆-tag, HNH was subsequently purified by HiPrep 16/60 Sephacryl 100 S-100 HR gel filtration chromatography. Crystals were obtained with sitting drop vapor diffusion at room temperature with 48 mg/mL HNH 1:1 with the Molecular Dimensions Morpheus I Screen condition E4 (0.1 M mixture of [imidazole and MES] pH 6.5, 25% (v/v) mixture of [2-methyl-2,4-pentanediol, PEG1000, and PEG3350], and 0.3 M mixture of [diethylene glycol, triethylene glycol, tetraethylene glycol, and pentaethlyene glycol]). Diffraction data were collected on a Rigaku MicroMax-003i sealed tube X-ray generator with a Saturn 944 HG CCD detector and processed and scaled using XDS^{64 (link)} and Aimless in the CCP4 program suite.^{65 (link)} The HNH domain from full-length S. pyogenes Cas9 was used for molecular replacement (PDB: 4UN3)^{5 (link)} with Phaser in the PHENIX software package.^{66 (link)} Iterative rounds of manual building in Coot^{67 (link)} and refinement in PHENIX yielded the final HNH domain structure.

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East K.W., Newton J.C., Morzan U.N., Narkhede Y., Acharya A., Skeens E., Jogl G., Batista V.S., Palermo G, & Lisi G.P. (2020). Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics. Journal of the American Chemical Society, 142(3), 1348-1358.

Publication 2020

Saturn 944 HG

Cleavage Diethylene glycol Diffusion Gel filtration chromatography Glycol His6 tag Imidazole Molecular condition Peg1000 Peg3350 Pyogenes s Tetraethylene glycol Triethylene glycol X ray

Corresponding Organization : University of California, Riverside

Other organizations : Yale University

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Variable analysis

independent variables

Concentration of HNH protein used for crystallization (48 mg/mL)

dependent variables

Successful crystallization of HNH protein
Diffraction data collected

control variables

Temperature (room temperature)
Crystallization method (sitting drop vapor diffusion)
Crystallization conditions (0.1 M mixture of [imidazole and MES] pH 6.5, 25% (v/v) mixture of [2-methyl-2,4-pentanediol, PEG1000, and PEG3350], and 0.3 M mixture of [diethylene glycol, triethylene glycol, tetraethylene glycol, and pentaethlyene glycol])

controls

Positive control: Crystal structure of the HNH domain from full-length S. pyogenes Cas9 (PDB: 4UN3) used for molecular replacement

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