Protein production and purification

The SARS-CoV-2 nsp12 (GenBank: MN908947) gene was cloned into a modified pET-22b vector, with the C-terminus possessing a 10× His-tag (primer information in Supplementary Table 5). The plasmids were transformed into E. coli BL21 (DE3), and the transformed cells were cultured at 37 °C in LB media containing 100 mg/L ampicillin. After the OD600 reached 0.8, the culture was cooled to 16 °C and supplemented with 0.5 mM IPTG. After overnight induction, the cells were harvested through centrifugation, and the pellets were resuspended in buffer 1 (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM MgCl₂, 10% glycerol) and homogenized with an ultra-high-pressure cell disrupter at 4 °C. The insoluble material was removed through centrifugation at 20,000 × g for 50 min. The fusion protein was incubated with Ni-NTA resin (GE Healthcare, USA) at 4 °C for 1 hour. After eluted by buffer 1 supplemented with 300 mM imidazole, the target protein was further purified by passage through a Hitrap Q ion-exchange column (GE Healthcare, USA) with buffer A (20 mM Tris-HCl, pH 8.0, 4 mM MgCl₂, 10% glycerol, 4 mM DTT) and buffer B (20 mM Tris-HCl, pH 8.0, 1 M NaCl, 4 mM MgCl₂, 10% glycerol, 4 mM DTT). Then it was loaded onto a Superdex 200 10/300 Increase column (GE Healthcare, USA) with buffer 2 (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 4 mM MgCl₂). Purified nsp12 was concentrated to 4.8 mg/mL and stored at 4 °C.

The SARS-CoV-2 nsp13 (GenBank: MN908947) gene was inserted into the modified pET-28a vector with a 6× His-tag attached at its N-terminus (primer information in Supplementary Table 5). E. coli BL21 (DE3) cells were then transformed by the introduction of this plasmid. Bacteria was induced when OD600 was 0.6–0.8 with 0.2 mM IPTG after cultured in LB medium at 37 °C. After grown at 16 °C for 16–18 h, cells were harvested and resuspended in buffer 3 (20 mM HEPES, pH 7.0, 150 mM NaCl, 4 mM MgCl₂, 10% glycerol). The cell pellets were centrifuged at 20,000 × g for 40 min after lysed by high-pressure homogenization and sonication. After purification by Ni-NTA (Novagen, USA) affinity chromatography, the protein eluted with 200 mM imidazole. The eluate was then further purified by Hitrap S ion-exchange column (GE Healthcare, USA) in a buffer containing 20 mM HEPES, pH 7.0, 50–300 mM NaCl gradient, 2 mM DTT, before loading onto a Superdex 200 10/300 Increase column (GE Healthcare, USA) in the buffer 4 (20 mM HEPES, pH 7.0, 150 mM NaCl, 4 mM MgCl₂, 4 mM DTT). Purified nsp13 was concentrated to 4 mg/mL and stored at 4 °C.

Full-length SARS-CoV-2 nsp7 and nsp8 were co-expressed in E. coli BL21 (DE3) cells as a no-tagged protein and a 6 × His-SUMO fusion protein, respectively (primer information in Supplementary Table 5). After purification by Ni-NTA (Novagen, USA) affinity chromatography, the nsp7–nsp8 complex was eluted through on-column tag cleavage by ULP protease. The eluate was further purified by Hitrap Q ion-exchange column (GE Healthcare, USA) and a Superdex 200 10/300 Increase column (GE Healthcare, USA) in a buffer 5 (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 4 mM MgCl₂).

Assembly of mini RTC

First, for assembling stable nsp12–nsp7–nsp8 complex, purified nsp12 with 4.8 mg/mL was incubated with nsp7 and nsp8 at 4 °C for 3h , at a molar ratio of 1:2:2 in a buffer containing 20 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 4 mM MgCl₂. Then the mixture was purification by mono Q 5/50 ion-exchange chromatography (GE Healthcare, USA), and got a stable nsp12–nsp7–nsp8 complex. Annealed RNA scaffold (Fig. 1a) was added to the dialyzed nsp12–nsp7–nsp8 complex at a molar ratio of 1:1 and incubated for 30 min at 25 °C. Then the mixture was incubated with the pretreated nsp13 which was incubated with GDP•BeF₄⁻ for 30 min at 25 °C to assemble mini RTC.

Nucleic acid unwinding assay

The nucleic acid unwinding assays were performed as previously reported⁹. Briefly, dsDNA (5′-AATGTCTGACGTAAAGCCTCTAAAATGTCTG-3′-BHQ, CY3-5′-CAGACATTTTAGAGG-3′) was used where the excitation wavelength was set to 547 nm and emission wavelength was set to 562 nm to detect fluorescence of CY3. 200 nM Nsp13 (final concentration) was added to the reaction buffer (50 mM HEPES, pH 7.0, 150 mM NaCl, 4 mM MgCl₂, 0.5 mM EDTA, and 0.1 mg/ml BSA) to incubate with dsDNA and 20 µM trap ssDNA for 5 min. Subsequently, 2 mM ATP (final concentration) was added to initiate the helicase activity, and the fluorescence value was recorded by Perkin-Elmer Enspire plate reader (Perkin-Elmer, USA).

Cryo-EM sample preparation and data collection

In total, 3 μL of protein solution at 3 mg/mL (added with 0.025% DDM) was applied onto an H₂/O₂ glow-discharged, 200-mesh Quantifoil R0.6/1.0 grid (Quantifoil, Micro Tools GmbH, Germany). The grid was then blotted for 3.0 s with a blot force of 0 at 8 °C and 100% humidity and plunge-frozen in liquid ethane using a Vitrobot (Thermo Fisher Scientific, USA). Cryo-EM data were collected with a 300 keV Titan Krios electron microscope (Thermo Fisher Scientific, USA) and a K3 direct electron detector (Gatan, USA). Images were recorded at ×29,000 magnification and calibrated super-resolution pixel size 0.82 Å per pixel. The exposure time was set to 2 s with a total accumulated dose of 60 electrons per Ų. All images were automatically recorded using SerialEM. A total of 4107 images were collected with a defocus range from −2.0 μm to −1.0 μm. Statistics for data collection and refinement are in Supplementary Table 1.

Cryo-EM image processing

All dose-fractioned images were motion-corrected and dose-weighted by MotionCorr2²¹ software, and their contrast transfer functions were estimated by ctffind4²². A total of 384,727 particles were auto-picked using blob picker and extracted with a box size of 440 pixels in cryoSPARC²³. The following 2D, 3D classifications, and refinements were all performed in cryoSPARC. 166,942 particles were selected after two rounds of 2D classification based on the complex integrality. This particle set was used to do Ab-Initio reconstruction in six classes, which were then used as 3D volume templates for heterogeneous refinement, with 109,912 particles converged into one nsp12-nsp7–nsp8–nsp13–RNA complex class. Next, these particles were imported into RELION 3.03²⁴ to perform local classification to obtain two different conformations. These two conformations are used as an initial model for heterogeneous refinement in cryoSPARC and particles are classified into two particle sets. Finally, non-uniform refinement is applied to the particle sets and result in two maps in final resolution 2.98 Å and 3.84 Å, respectively.

Model building and refinement

To solve the structure of the SARS-CoV-2 mini RTC complex, the structure of the SARS-CoV-2 nsp12 and nsp7-8 complex (PDB: 7BTF) and SARS-CoV-2 nsp13 (PDB: 6ZSL) were individually placed and rigid-body fitted into the cryo-EM map using UCSF Chimera²⁵. The model was manually built in Coot²⁶ with the guidance of the cryo-EM map and in combination with real space refinement using Phenix²⁷. The data validation statistics are shown in Supplementary Table 1.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.