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. 2019 Aug 9;8:e49110. doi: 10.7554/eLife.49110

Figure 1. Overall structure of the LbCas12a-crRNA-AcrVA4 complex.

(A) Schematic representation of Cas12a activity. (B) Unrooted maximum likelihood phylogenetic tree of Type V-A CRISPR-Cas12a. Species known to be susceptible or unsusceptible to phage-derived AcrVA4 are highlighted. The triangle denotes collapsed branches of Cas12b-e. (C) Schematic representation of LbCas12a and the mature crRNA modeled within the cryo-EM structures. (D) Two views of the LbCas12a-crRNA complex (cartoon) bound to AcrVA4 (surface) shown related by a 180° rotation. The color scheme for the crRNA, LbCas12a, and AcrVA4 in panels B, C, and D are used throughout the manuscript.

Figure 1.

Figure 1—figure supplement 1. Cryo-EM data collection and 3D reconstruction.

Figure 1—figure supplement 1.

(A) Electron microscopy analysis pipeline. From 3350 drift-corrected micrographs,~800,000 particles were picked and used for 2D classification. By 2D-based manual screening, 324,336 good particles were selected for 3D classification into four classes. From the classes that show a monomeric Cas12a architecture (Class 2 and Class 3), 156,979 particles were further used for refinement to generate reconstruction State I. From the class that shows a dimeric Cas12a architecture (Class 4), 79,786 particles were used for refinement to generate reconstruction State II. State I and State II were then independently refined to 3.0 Å and 5.0 Å, respectively. (B) The density maps for both states, colored by local resolution as calculated in Cryosparc. The resolution ranges from 2.5 Å to 4.5 Å in State I and 4.5 Å to 7.5 Å in State II. (C) Euler angle distribution of the refined particles belonging to State I (top) and State II (bottom). (D) Gold-standard Fourier shell correlation (GSFSC) curve calculated using two independent half-maps for State I (top) and State II (bottom).

Figure 1—figure supplement 2. Cryo-EM density for State I.

Figure 1—figure supplement 2.

(top) Segmented cryo EM density map for State I illustrating the LbCas12a protein chain (gray), the crRNA (blue) and the CBD of AcrVA4 (red) (map contoured to 10 σ). (A) Detailed views of cryo EM density describing the LbCas12a WED domain (yellow), RuvC domain (blue), and bridge helix (green) (maps contoured to 10 σ). (B) Detailed view of the cryo EM density describing the LbCas12a bound crRNA (map contoured to 15 σ). (C) Detailed view of the cryo EM density describing the overall fold of the AcrVA4 CBD (top) and an example of the high-resolution that allowed for side chain assignment in the map (bottom).

Figure 1—figure supplement 3. Structurally similar proteins to AcrVA4 CBD as determined by Dali search.

Figure 1—figure supplement 3.

Cartoon representation of (A) AcrVA4 (red), (B) Pyrobaculum spherical virus protein (PDB code: 2X5C, gray), (C) TrmB archaeal transcriptional regulator protein (PDB code: 3QPH, blue), and (D) Argonaute protein PAZ domain (PDB code: 4Z4H, yellow).