Materials and Methods

Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM

MATERIALS AND METHODSSeCsm2 and SeCsm3 recombinant plasmid construction and expressionThe coding region for the csm2 and csm3 genes were PCR-amplified from extracted S. epidermidis RP62a genomic DNA using PCR primers for csm2 (Csm2-F and Csm2-R) and csm3 (Csm3-F and Csm3-R) (Supplementary Table S1). PCR-amplified genes were inserted into the pET21-derived expression plasmid pMCSG7 (40) through the ligation-independent cloning (LIC) region. Utilizing the LIC region introduces an in-frame 6X Histidine tag and a tobacco etch virus (TEV) cleavage site at the N-terminus of the protein. The constructs were confirmed by DNA sequencing (ACGT Inc., Germantown, MD) and subsequently transformed into Rosetta competent cells (EMD Millipore) for protein expression and purification. Both constructs were grown in Terrific Broth (TB) media supplemented with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol at 37°C to an OD600 of 0.8. Cells were cooled on ice for 1 h to reduce leaky gene expression before induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 18 h at 16°C. Cells were harvested by centrifugation at 5,500 × g for 15 min at 4°C. Cell paste was then flash frozen in liquid nitrogen and stored at −80°C until purification.Expression of selenomethionine substituted SeCsm2 and SeCsm3 was performed by growth of a 5 ml pre-culture in Luria Broth (LB) rich media grown for 8 h at 37°C. A 10 ml M9 minimal media starter culture was inoculated with 1:1000 pre-culture and grown overnight at 37°C. One liter of M9 minimal media cultures were inoculated with 1:100 M9 starter and grown to an OD600 of 0.8. Cells were cooled on ice for 45 min before adding 100 mg lysine, 100 mg threonine, 100 mg phenylalanine, 50 mg leucine, 50 mg isoleucine, 50 mg valine and 50 mg l(+)-selenomethionine (ACROS Organics). After 15 min, cells were induced with 0.5 mM IPTG for 18 h at 16°C. Cells were harvested and frozen following the same protocol as for the native proteins.Recombinant SeCsm2 protein purificationPellets were thawed on ice for 30 min before adding 1:1 volume of resuspension buffer (100 mM Tris pH 8, 1M NaCl, 20 mM imidazole). Resuspended cells were incubated with 50 μM lysozyme, 0.12% Brij 58, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM benzamidine on a nutating mixer for 30 minutes at 4°C. Cells were lysed using a Misonix S-4000 sonicator with a 1/2" horn (Amplitude 20, 5 s on, 10 s off, 5 min total on-time). Cell lysate was clarified through ultracentrifugation at 160,000 × g for 45 min at 4°C in a Ti-70 rotor (Beckman-Coulter). Insoluble cell debris was removed and supernatant was filtered through a 0.2 μm polyethersulfone (PES) membrane bottle-top filter. Filtered lysate was loaded onto a column containing 10 ml nickel nitrilotriacetic acid (Ni-NTA) Superflow resin (Qiagen) equilibrated with buffer 1 (50 mM Tris pH 8, 500 mM NaCl, 10 mM imidazole). The column was washed with 500 ml buffer 1, followed by protein elution in 10 ml fractions of elution buffer 1 (50 mM Tris pH 8, 500 mM NaCl, 250 mM imidazole). Elution fractions containing protein were dialyzed overnight at 4°C against 1.5 l dialysis buffer 1 (50 mM Tris pH 8, 500 mM NaCl). Dialyzed protein was concentrated using a Vivaspin 20 3 kDa molecular weight cutoff (MWCO) concentrator (GE Healthcare) to 2 ml. The protein was then diluted to 100 mM NaCl immediately before loading onto a column packed with 10 ml High-S cation-exchange resin (Bio-Rad) attached to a Gilson Minipuls 3 peristaltic pump at 1.2 ml/min to reduce the likelihood of precipitation in low salt. The column was washed with 50 ml of low salt buffer (50 mM Tris pH 8, 100 mM NaCl), followed by a 100 ml linear gradient from 100 mM to 1 M NaCl. Protein-containing fractions were pooled and dialyzed overnight at 4°C against 1.5 l dialysis buffer 1. Finally, the protein was concentrated to 0.7–1.2 mg/ml for crystallization experiments. Selenomethionine-labeled SeCsm2 (SeMet–SeCsm2) was purified using the same protocol.Recombinant SeCsm3 protein purificationPellets were thawed on ice for 30 min before adding 1:1 volume of no salt buffer (50 mM Tris pH 8, 20 mM imidazole). Resuspended cells were incubated with 50 μM lysozyme, 0.12% Brij 58, 1 mM PMSF and 1 mM benzamidine on a nutating mixer for 30 min at 4°C. Additionally, 10 mM MgCl2, 2 mM CaCl2 and 16 μM bovine pancreas deoxyribonuclease I (Sigma Aldrich) were added and allowed to stir for 15 min at room temperature. Following this incubation, NaCl was added to a final concentration of 500 mM. Cells were further lysed using a Misonix S-4000 sonicator with a 1/2" horn (Amplitude 20, 5 s on, 10 s off, 5 min total on-time). Insoluble cell debris was removed by ultracentrifugation at 160,000 × g for 45 min at 4°C in a Ti-70 rotor (Beckman-Coulter). To remove unbound nucleic acids, a 5% solution of polyethyleneimine (PEI) pH 7.9 was added dropwise to a final concentration of 0.2% into the supernatant while stirring at 4°C (41). The solution was stirred for 5 min before removing the precipitated nucleic acids through centrifugation at 18,000 × g for 30 min in a FA-45-6-30 fixed-angle rotor (Eppendorf). The clarified lysate was filtered through a 0.2 μm PES membrane bottle-top filter. Filtered lysate was loaded onto a 10 ml Ni-NTA Superflow resin column equilibrated with buffer 1. The column was washed with 200 ml buffer 1, followed by 400 ml buffer 2 (50 mM Tris pH 8, 200 mM NaCl, 10 mM imidazole). Protein was eluted in 10 ml fractions with elution buffer 2 (50 mM Tris pH 8, 200 mM NaCl, 250 mM imidazole). Protein-containing fractions were incubated with 1:10 molar concentration of TEV protease while dialyzing overnight at 4°C against 1.5 l dialysis buffer 2 (50 mM Tris pH 8, 100 mM NaCl, 10 mM imidazole). The dialysis bag was transferred to fresh 1.5 l dialysis buffer 2 and allowed to equilibrate again overnight at 4°C. Dialyzed, cleaved protein was subjected to another round of affinity chromatography to remove the cleaved His tag, uncleaved protein, and His-tagged TEV protease. The Ni-NTA column flow through was loaded onto a 10 ml High-S cation exchange resin column equilibrated with 50 mM Tris pH 8, 100 mM NaCl at 0.4 ml/min. Nucleic acids bind to the resin, allowing the nucleic acid-free protein to be collected in the flow through. Nucleic acid-free SeCsm3 was concentrated with a Vivaspin 20 10 kDa MWCO concentrator to 4–7 mg/ml to be used for crystallization experiments. SeMet–SeCsm3 was purified using the same protocol.Crystallization conditions of SeCsm2 and SeCsm3Crystallization trials of native and SeMet-derived SeCsm2 and SeCsm3 were conducted using a variety of commercial screens and utilizing both the hanging drop and sitting drop vapor diffusion methods. The best diffracting SeCsm2 and SeMet-SeCsm2 crystals were set up at 4°C in VDX hanging drop vapor diffusion plates (Hampton) and grown at 10°C in 100 mM Tris pH 7, 8% ethanol at 1.2 mg/ml. Crystals formed after 1–2 days of incubation. Crystals were cryo-protected by supplementing the crystallization condition with 20% ethylene glycol and 10% ethanol. SeMet–SeCsm3 crystals yielding the best diffraction data were set up at room temperature in Cryschem sitting drop vapor diffusion plates (Hampton) and grown at 14°C in 20% polyethylene glycol (PEG) 8000, 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 6.5, 200 mM calcium acetate at 6.8 mg/ml. Crystals formed after 24 h of incubation. SeMet–SeCsm3 crystals were cryo-protected by supplementing the crystallization condition with 20% ethylene glycol. For heavy atom derivatization, SeMet–SeCsm3 crystals were soaked for 2 min in the cryo-protectant supplemented with 10 mM samarium (III) chloride before freezing.SeCsm2 structure determinationX-ray diffraction data were collected from native SeCsm2 and SeMet-SeCsm2 crystals at 100 K at the Life Sciences Collaborative Access Team (LS-CAT) beamlines at the Advanced Photon Source, Argonne National Laboratory, using a Rayonix MX300 CCD detector. All crystallographic data were processed with XDS (42) and Aimless (43). Positions of three Se atoms were obtained with the program Shake-and-Bake (44). A Single Anomalous Dispersion (SAD) electron density map was initially obtained using SHARP (45). The map showed clearly the fold of the protein, but the data were limited to 3.2 Å resolution. An experimental electron density map including both the SeMet data and a higher resolution native data set was calculated with SHARP (45) and was of better quality. The combined SeMet/native map allowed tracing of most of the molecule with the aid of Coot (46) followed by iterative model building and refinement with BUSTER (47), Phenix (48,49), and Refmac5 (50). The final model spans most of the molecule, aside from one short disordered loop (residues 29–36) and two amino acids missing at the C-terminus, and includes nine amino acids that form part of the His-tag used for purification. The stereochemistry of the model was validated with MolProbity (51) and Coot (46). The final model has an Rwork/Rfree of 24.47%/29.42% to 2.75 Å resolution with excellent stereochemistry. X-ray crystallographic statistics for the structure are summarized in Supplementary Table S2.SeCsm3 structure determinationX-ray diffraction data were collected from SeMet–SeCsm3 crystals at 100 K at LS-CAT beamlines at the Advanced Photon Source, Argonne National Laboratory, using a Dectris Eiger 9M detector. Molecular Replacement calculations failed to give a solution and hence data from SeMet crystals soaked in 10 mM samarium (III) chloride were collected with the incident beam tuned to 11.5 keV (1.0781 Å) and 6.724 keV (1.8439 Å) for experimental phasing. Diffraction data were processed using XDS (42) and Aimless (43). An experimental electron density map was calculated using the automated pipeline CRANK-2 (52) in CCP4 (53) using the 1.8439 Å wavelength data set. Initial model building was performed with CRANK-2 (52) in CCP4 (53) followed by multiple iterations of automated model building using Buccaneer (54) and manual model editing using Coot (46). The model was refined against the 1.0781 Å data set using Refmac5 (50). The final model contains two molecules in the asymmetric unit (root-means-square-deviation RMSD of 0.15 Å) and spans most of the molecule (A monomer 2–20, 32–65, 74–124 and 137–214, B monomer 0–20, 31–65, 75–124, 137–214), with the missing amino acids residing in three disordered loops. The stereochemistry of the model was validated with MolProbity (51) and Coot (46). The final model has an Rwork/Rfree of 23.1%/26.7% to 2.40 Å resolution with excellent stereochemistry. X-ray crystallographic statistics for the structure are summarized in Supplementary Table S3. S. epidermidis RP62a Cas10–Csm complex purification S. epidermidis RP62a harboring pcrispr with Cas10 N-terminally His-tagged was generously provided by Dr. Asma Hatoum-Aslan at the University of Alabama (26). Strains were grown in BBL brain heart infusion media (BD). The media was supplemented with neomycin (15 μg/ml) and chloramphenicol (10 μg/ml). Cultures (1 l culture per 2 l flask) were grown for 24 h at 37°C. Cells were harvested, frozen at −80°C, and stored until purification. Cell pellets from 6 l of culture volume were thawed at 4°C for 30 min and resuspended in 30 ml of resuspension buffer (30 mM MgCl2, 35 μg/ml lysostaphin Ambi Products) supplemented with PMSF and benzamidine (1 mM final concentration). Cells were incubated at 37°C for 1 h, mixing every 20 min. Cell lysates were subsequently diluted with 1/3 lysate volume of lysis buffer (200 mM NaH2PO4 pH 7.4, 2 M NaCl, 80 mM imidazole, 0.4% Triton X-100, 2 mM PMSF, 2 mM benzamidine). Sonication was performed on the diluted lysates on ice (Amplitude 25, 5 s on, 10 s off, 10 min total on-time Misonix S-4000, 1/2" horn). Sonicated lysate was spun at 18,000 × g for 45 min at 4°C in a FA-45-6-30 fixed-angle rotor. The insoluble cell debris was removed and spun again at 18,000 × g for 30 min. The remaining cell debris was discarded and the clarified lysate was filtered through a 0.2 μm PES membrane bottle-top filter. The filtered, clarified lysate was loaded onto a column packed with Ni-NTA Superflow resin (1 ml slurry per 1 l culture volume) equilibrated with equilibration buffer (50 mM NaH2PO4 pH 7.4, 500 mM NaCl, 10 mM imidazole). The lysate was passed over the resin twice before washing with 600 ml wash buffer (50 mM NaH2PO4 pH 7.4, 500 mM NaCl, 20 mM imidazole). Protein was eluted from the column with 25 ml elution buffer A (50 mM NaH2PO4 pH 7.4, 500 mM NaCl, 250 mM imidazole) in 5 ml fractions. Protein-containing fractions were pooled and dialyzed against 1.5 l dialysis buffer A (20 mM Tris pH 8, 200 mM NaCl) at 4°C overnight. Dialyzed complex was diluted to 100 mM NaCl immediately before loading onto a 10 ml column packed with Q Sepharose Fast Flow resin (GE Healthcare) equilibrated with buffer B (20 mM Tris pH 8, 100 mM NaCl) running at 1.4 ml/min. The column was washed with 25 ml of buffer B, followed by a 100 ml linear gradient from 100 mM to 2 M NaCl. Fractions of interest were pooled and dialyzed against 1.5 l dialysis buffer B (20 mM Tris pH 8, 500 mM NaCl) at 4°C overnight. Dialyzed complex was then concentrated to ∼300 μl and loaded onto a Superdex 200 10/300 GL (GE Healthcare) equilibrated with dialysis buffer B. The non-aggregated complex peak was collected. Peak fractions are stored at 4°C for up to 1 month without any noticeable degradation as assayed by SDS-PAGE.Mass spectrometry analysis of the crRNAcrRNAs were isolated by multiple rounds of 1:1 phenol-chloroform extractions from the purified effector complex gel filtration peak fractions used in the EM structural studies. Following extraction, the crRNAs were ethanol precipitated with 1/10 volume 3 M sodium acetate, pH 5.2 and 3 volumes 100% ethanol and stored at −20°C. Samples were spun down and washed three times with 80% ethanol to remove excess salt. A final spin was performed, the ethanol was removed, and the crRNAs were dried. Once dry, the lyophilized pellets were resuspended in ddH2O and submitted to the Integrated Molecular Structure Education and Research Center (IMSERC) at Northwestern University for mass spectrometry. Samples were run on an Agilent 6210A LC-TOF High Resolution Time of Flight Mass Spectrometer using ElectroSpray Ionization (ESI), connected to an Agilent 1200 series HPLC. Samples were analyzed using a Direct Loop injection, with water/acetonitrile (60/40) as carrier solvent. MassHunter Workstation Data Acquisition software was used for instrument operation and MassHunter Qualitative Analysis software for data analysis and processing. in vitro target RNA cleavage assay in vitro time-course RNA cleavage assays were performed by incubating 100 nM SeCas10–Csm complex with 50 nM 5′-fluorescein amidite (FAM)-labeled target RNA complementary to the Spacer 1 sequence (5′-FAM-AGCCUGACUGAUGAUUUAUAUACUUCGGCAUACGUUCCAG-3′; Integrated DNA Technologies IDT) in a 30 μl reaction. Reactions were performed in 20 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, 2 mM DTT and 3% glycerol. Control reactions replaced the MgCl2 with 10 mM ethylenediaminetetraacetic acid (EDTA) to abolish cleavage activity. Reactions were stopped by addition of 2× formamide loading buffer (90% formamide, 0.025% SDS, 5 mM DTT), heated to 95°C for 5 min, analyzed on a 15% polyacrylamide denaturing 8 M urea gel, and visualized with a Typhoon 9400 scanner (GE Healthcare). In addition, cleavage assays under identical conditions but using a non-target RNA (bacterial RNase P) showed no cleavage, confirming that cleavage was not the result of contaminating nucleases.Negative stain electron microscopy sample preparation and data acquisitionCarbon-coated copper Gilder grids (300 mesh, Electron Microscopy Sciences EMS) were glow-discharged for 7 s at 10 W in a Solarus 950 plasma cleaner (Gatan). Glow-discharged grids were placed carbon side down onto a 30 μl drop of 30 μg/ml purified complex for 5 min at room temperature. Grids were subsequently transferred twice to 30 μl drops of 20 mM Tris pH 8, 500 mM NaCl for 1 min per drop, with blotting occurring after the second transfer. The grids were then transferred twice to 30 μl drops of 2% uranyl acetate solution for 30 seconds per drop, followed by blotting after the second transfer and finally allowing the grid to air dry. Once dry, the grids were imaged on a JEOL 1400 transmission electron microscope (TEM) operating at 120 keV. 175 micrographs were collected using Leginon (55) at a magnification of 40,431× using an UltraScan4000 CCD camera with a pixel size of 3.71 Å at the specimen level and using a defocus range of –2 μm to –3 μm. Micrographs were processed using RELION-2.1 (56,57). A small group of micrographs were selected for manually picking particles for initial 2D classification and the 2D classes were used for subsequent auto-picking of the entire dataset (∼385,000 particles). Auto-picked particles were subjected to successive rounds of 2D classification to remove improperly picked particles yielding ∼175,000 particles. The ∼175,000 particles formed two distinct subsets of defined 2D classes. The first subset contained ∼63,000 particles detailing a more linear complex, whereas the second subset of ∼112,000 particles represented a double-helical complex. Each subset was subjected to 3D classification. An input model for each subset was generated ab initio from a subset of these particles. The model best representing the 2D classes was chosen as the initial model for cryo-EM studies.Cryo-Electron microscopy sample preparation and data acquisitionCryo-EM grids (C-flat, 1.2/1.3, 400 mesh, Cu; EMS) were glow-discharged for 10 s at 15 mA in an easiGlow glow discharger (Pelco). Grids were prepared under >95% humidity at 4°C using a Vitrobot Mark IV (FEI ThermoFisher). A glow-discharged cryo grid was placed in the Vitrobot sample chamber, onto which a 3 μl drop of protein (∼0.2 mg/ml) was deposited. After waiting 5 s, the grid was blotted for 4 s with a blot force of +5 and immediately plunged into liquid ethane. The grid was transferred to a grid box for storage until data collection. Leginon (55) was used for automated EM image collection. Micrographs were collected using a JEOL 3200FS TEM operating at 300 keV equipped with an in-column energy filter (Omega filter) and a K2 Summit direct electron detector (Gatan). A magnification of 40,323 × was used, for a pixel size of 1.24 Å at the specimen level and a defocus range of –2 μm to –3.5 μm. Movies were collected in counting mode with a total dose of ∼37.5 e−/Å2, fractionated into 0.2 s frames for a total of 8 s, corresponding to 40 frames per movie. A total of 1,037 movies were collected in this manner.Cryo-EM image processing and model buildingCryo-EM movies were processed using RELION-3.0 (57–59). Motion correction was performed using the program MotionCor2 (60) and dose-weighted according to the relevant radiation damage curves (61) within RELION-3.0 (58) to allow for downstream post-processing. The Contrast Transfer Function (CTF) was estimated using CTFfind4 (62) on motion-corrected micrographs. 277,413 particles were auto-picked from templates generated from manual 2D classification. Auto-picked particles (277,413) were subjected to 3D classification, with the best class containing 107,620 particles. Additional RELION-3.0 post processing steps included per-particle CTF estimation and particle polishing to further improve the resolution. The particle polishing step showed a significant improvement on the final calculated volume, which had a 5.2 Å resolution according to the Fourier Shell Correlation (FSC) criterion (63). Initial inspection of the map showed that the quality was uneven, with the two extremes of the molecule at lower resolution than the central stem. The map was segmented into two pieces, the major stem (5× SeCsm3 and 1× SeCsm4), and the rest of the volume (putatively SeCas10), to use in multi-body refinement using RELION-3.0 (59). Multi-body refinement did not improve the resolution of the stem, but did improve slightly the volume around the SeCas10 domain.To position the SeCsm3 crystal structure in the cryo-EM map, a 6 dimensional search was done using the program Essens (64), which identified five copies of SeCsm3 along the stem. The positions corresponded to the same locations identified visually. Essens and visual searches using SeCsm2 as a template found no matches, indicating SeCsm2 was not part of the complex. Further visual inspection of the map showed the presence of an additional RRM motif at the base of the stem. Superposition of SeCsm3 on the RRM motif showed good agreement for the RRM region, but not for other regions, suggesting that the region corresponded to an RRM-containing protein distinct from SeCsm3. This was assigned as SeCsm4. The remaining density was assigned as SeCas10 based on its position and size. No density corresponding to SeCsm5 could be identified. Once all five SeCsm3 molecules had been identified, their position was refined using jigglefit (65) in Coot (46). The models showed excellent fit in most areas, but some loops and secondary structure elements needed adjustment to fit the density. To improve the placing of the crystal structures, the monomers were fit into the map using the MDFF routines (66) that are part of NAMD (67). No further refinement of the models was done. Unless noted, the figures were made using the crystal structures fit into the map.Model superpositions, comparisons, and figuresComparisons of the different models were all done using Coot (46) and Chimera (68). When superposing structures from different organisms, the superposition was based on secondary structure matching (SSM) as implemented in Coot (46) and Chimera (68).Figures were drawn with Pymol (69) and Chimera (68). Conservation data were generated using the ConSurf server (70,71) and visualized in Pymol (69). Electrostatic potential calculations were done with APBS (72). Homology models were generated using the Phyre2 server (73) and visualized in Pymol (69) and Chimera (68).

Article TitleStructural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM

Abstract

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession numbers 6NBT and 6NBU. The electron density map for the cryo-EM structure has been deposited in the EMDataBank under accession number EMD-0442.


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