MATERIALS AND METHODSPlasmid constructs and protein purificationDNA sequences of AcrIF4, AcrIF7 and AcrIF14 were ordered as gBlocks from Integrated DNA Technologies, Inc., which were cloned individually into pET His6 Sumo TEV LIC cloning vectors (1S) (Addgene # 29659) using the Gibson Assembly® Master Mix (NEB, Cat. # E2611S). After sequence verification, these plasmids were transformed into BL21 (DE3) cells for expression in Terrific Broth medium. Protein expression was induced by 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16°C overnight. Cell pellets were resuspended in lysis buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl, 5% glycerol, 5 mM β-meracptoethanol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and disrupted by sonication. After centrifugation, the supernatant was loaded in a HisTrap HP column. AcrF proteins were eluted with a stepwise gradient of 1.0 M imidazole, digested with TEV protease overnight for removal of 6XHis and SUMO-tags, and purified with ion-exchange chromatography using either a Heparin HiTrap Q HP or Heparin HiTrap SP HP depending on protein isoelectric point (pI). AcrIF proteins were then concentrated and further purified over a Superdex 200 column (GE Healthcare) in a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 5% glycerol and 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP).For purification of the PA14 Csy complex, the pCsy_complex plasmid (Addgene ID# 89232) and pCRISPR_DMS3g24 (Addgene ID # 89244) were co-transformed into BL21 (DE3) cells for expression. Cell pellets were resuspended in lysis buffer containing 50 mM HEPES (pH 7.5), 300 mM KCl, 5% glycerol, 0.2 mM PMSF, 1 mM TCEP and cOmplete™ protease inhibitor (Roche, 04693132001), and disrupted by sonication. Intact Csy complex was purified by a HisTrap HP column (GE Healthcare) as previously described for the AcrIF proteins, and the buffer was exchanged overnight into a buffer containing 50 mM HEPES (pH 7.5), 150 mM KCl, 5% glycerol, and 1 mM TCEP. After ion-exchange chromatography using a Heparin HiTrap Q column (GE Healthcare), the Csy complex was further purified using a Superdex 200 column (GE Healthcare) equilibrated in a buffer containing 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol, and 1 mM TCEP.Complex assemblyTo assemble Csy-AcrIF complexes, purified Csy was incubated with AcrIF proteins at a molar ratio of 1:10 for 1 h on ice, and then subjected to size exclusion chromatography over a Superdex 200 column (GE Healthcare) equilibrated in buffer containing 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol and 1 mM TCEP, followed by SDS-PAGE analysis of the elution fractions.To test whether AcrIF4, AcrIF7 and AcrIF14 can interact with the Csy complex simultaneously, Csy, AcrIF4, AcrIF7 and AcrIF14 were incubated at molar ratio of 1:3:3:3 for 1 h on ice, and then subjected to size exclusion chromatography over a Superdex 200 column (GE Healthcare) equilibrated in buffer containing 50 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol and 1 mM TCEP, followed by SDS-PAGE analysis of the elution fractions.Electromobility Shift Assays (EMSA)A dsDNA Substrate was prepared by mixing two complementary ssDNAs purchased from IDT. The target (5′-CAGGTAGACGCGGACATCAAGCCCGCCGTGAACAGGTAGACGCGGACATCAAGCCCGCCGTGAACAGGTAGACGCGGACATCAAGCCCG-3) and non-target strands (3′-GTCCATCTGCGCCTGTAGTTCGGGCGGCACTTGTCCATCTGCGCCTGTAGTTCGGGCGGCACTTGTCCATCTGCGCCTGTAGTTCGGGC-5′) were mixed and then denatured at 95°C for 5 minutes and then allowed to cool to room temperature before use in binding assays.Binding assays were performed with 400 nM Csy and increasing concentrations of AcrIF4 or AcrIF7 (0.4, 4.0, 8.0, 16, 40 and 80 μM) in reaction buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 1 mM TCEP, 5% glycerol, 2 mM MgCl2). Csy and inhibitors were first incubated for 1 h on ice before addition of dsDNA to a final concentration of 100 nM. Samples were then heated to 37°C for 30 min with mild agitation. Samples were then removed from heat, and reaction products were run on native 6% polyacrylamide TBE gels (DNA retardation gels, ThermoFisher). Gels were stained with SYBR Green Nucleic Acid Stain (ThermoFisher) and imaged with a GE Healthcare ImageQuant LAS 4000.Electron microscopyAliquots of 3 μl Csy-AcrIF4, Csy-AcrIF7 and Csy-AcrIF14 at 0.5 mg/ml were applied to glow-discharged UltrAuFoil holey gold grids (R1.2/1.3, 300 mesh). The grids were blotted for 2.5 s and plunged into liquid ethane using a ThermoFisher Scientific Mark IV Vitrobot. Cryo-EM data were collected with a Titan Krios microscope (FEI) operated at 300 kV and images were collected using Leginon (25) at a nominal magnification of 81,000x (resulting in a calibrated physical pixel size of 1.05 Å/pixel) with a defocus range of 1.2–2.5 μm. The images were recorded on a K3 electron direct detector in super-resolution mode at the end of a GIF-Quantum energy filter operated with a slit width of 20 eV. A dose rate of 20 electrons per pixel per second and an exposure time of 3.12 s were used, generating 40 movie frames with a total dose of ∼54 electrons/Å2. Statistics for cryo-EM data are listed in Table Table11.Table 1.Cryo-EM data collection, refinement and validation statisticsCsy-AcrIF4 (EMD-22582, PDB 7JZW)Csy-AcrIF7 (EMD-22583, PDB 7JZX)Csy-AcrIF14 (EMD-22585, PDB 7JZZ) Data collection and processing Magnification81 00081 00081 000Voltage (kV)300300300Electron exposure (e–/Å2)545454Defocus range (μm)1.5–2.51.5–2.51.5–2.5Pixel size (Å)1.051.051.05Symmetry imposed C1
C1 Initial particle images (no.)1 765,0621 627 8831 217 862Final particle images (no.)766 782502 177226 089Map resolution (Å)22.214.171.124FSC threshold0.1430.1430.143Map resolution range (Å)3–4.23.2–4.43–4.2 Refinement Initial model usedPDB 6NE0PDB 7JZWPDB 7JZWModel resolution (Å)126.96.36.199FSC threshold0.50.50.5Model resolution range (Å)3.2–503.4–503.2–50Map sharpening B factor (Å2)–112–129–86Model compositionNon-hydrogen atoms24 26623 94824 767Protein residues298629543092Nucleotides616161Ligands000 B factors (Å2)Protein28.4965.2648.40Nucleotide58.0175.1081.07R.m.s. deviationsBond lengths (Å)0.0100.0090.010Bond angles (°)0.9770.9210.980 Validation MolProbity score1.751.731.76Clashscore5.655.335.72Poor rotamers (%)0.130.400.00Ramachandran plotFavored (%)93.0092.9993.07Allowed (%)7.007.016.93Disallowed (%)0.000.000.00Open in a separate windowImage processingThe movie frames were imported to RELION-3 (26). Movie frames were aligned using MotionCor2 (27) with a binning factor of 2. Contrast transfer function (CTF) parameters were estimated using Gctf (28). A few thousand particles were auto-picked without template to generate 2D averages for subsequent template-based auto-picking. The auto-picked and extracted particle dataset were split into batches for 2D classifications, which were used to exclude false and bad particles that fall into 2D averages with poor features. Particles from different views were used to generate an initial model in cryoSPARC (29). 3D classification was then performed, followed by 3D refinement using particles in good 3D classes. Focused refinements around the inhibitors were further performed to improve the local resolutions.For the Csy-AcrIF4 dataset, 1 765 062 particles were auto-picked and extracted from the dose weighted micrographs. 1 123 229 particles were selected from 2D classification and used for 3D classification. 766 782 particles were selected from 3D classification and used for final 3D refinement. For the Csy-AcrIF7 dataset, 1 627 883 particles were auto-picked and extracted from the dose weighted micrographs. 989 921 particles were selected from 2D classification and used for 3D classification. 502 177 particles were selected from 3D classification and used for final 3D refinement. For the Csy-AcrIF14 dataset, 1 217 862 particles were auto-picked and extracted from the dose weighted micrographs. 1 174 148 particles were selected from 2D classification and used for 3D classification. 226 089 particles were selected from 3D classification and used for final 3D refinement. Statistics of cryo-EM image processing are summarized in Table Table11.Model building and refinement De novo model building of AcrIF4, AcrIF7 and AcrIF14 structures were performed manually in COOT (30). Secondary structure predictions by PSIPRED (31) were used to assist manual building. To build models of the Csy complex, each subunit of the structure of the Csy-target DNA complex (PDB:6NE0) was fitted into the maps as a rigid-body in UCSF Chimera (32) and manually adjusted in COOT. Refinement of the structure models against corresponding maps were performed using phenix.real_space_refine tool in Phenix (33).VisualizationFigures were generated using PyMOL and UCSF Chimera (32).
Article TitleStructural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14
Cryo-EM reconstructions of Csy-AcrIF4, Csy-AcrIF7 and Csy-AcrIF14 complexes have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-22582, EMD-22583 and EMD-22585, respectively. Coordinates for atomic models of Csy-AcrIF4, Csy-AcrIF7 and Csy-AcrIF14 complexes have been deposited in the Protein Data Bank under the accession numbers 7JZW, 7JZX and 7JZZ, respectively.