Open AccessArticle
Expression, Purification, Crystallization, and X-ray Structural Analysis of CRISPR-Associated Protein Cas6 from Methanocaldococcus jannaschii
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Ming-Chang Lee, Shih-Ting Tseng, Juan-Cheng Yang, Tung-Ju Hsieh, Shang-Chuen Wu, Shu-Min Kuan, Ming-Jen Chen, Ming-Chiu Chang, Chun-Chiu Wang, Hsiu-Lin Chen, Guor-Cheng Fang, Winn-Jung Huang, Tzu-Ping Ko and Yeh Chen
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Abstract
The CRISPR-associated protein 6, Cas6 protein, is an endoribonuclease that cleaves precursor CRISPR RNAs within the repeat sequence to release specific invader-targeting RNAs. Cas6 protein can recognize different sequences by their specific scaffold. To investigate its binding mode, we purified and crystallized a
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The CRISPR-associated protein 6, Cas6 protein, is an endoribonuclease that cleaves precursor CRISPR RNAs within the repeat sequence to release specific invader-targeting RNAs. Cas6 protein can recognize different sequences by their specific scaffold. To investigate its binding mode, we purified and crystallized a His-tagged Cas6 protein from
Methanocaldococcus jannaschii (MjCas6) using the sitting-drop vapor-diffusion method. The crystals diffracted to a resolution of 1.85 Å and belonged to monoclinic space group
C2, with unit-cell parameters
a = 200.84 Å,
b = 85.26 Å,
c = 100.06 Å, β = 118.47°. The crystals of MjCas6 contain four molecules in the asymmetric unit. The protein fold is similar to the other Cas6 homologues, such as
Pyrococcus furiosus Cas6, suggesting functional similarity. Moreover, in the C2 crystal the MjCas6 monomers formed a tandem array, which we hypothesize to possibly correlate with repetitive RNA precursors.
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