Atomic-Resolution Structure of SARS-CoV-2 Nucleocapsid Protein N-Terminal Domain

The nucleocapsid (N) protein is one of the four structural proteins of the SARS-CoV-2 virus and plays a crucial role in viral genome organization and, hence, replication and pathogenicity. The N-terminal domain (NNTD) binds to the genomic RNA and thus comprises a potential target for inhibitor and vaccine development. We determined the atomic-resolution structure of crystalline NNTD by integrating solid-state magic angle spinning (MAS) NMR and X-ray diffraction. Our combined approach provides atomic details of protein packing interfaces as well as information about flexible regions as the N- and C-termini and the functionally important RNA binding, β-hairpin loop. In addition, ultrafast (100 kHz) MAS 1H-detected experiments permitted the assignment of side-chain proton chemical shifts not available by other means. The present structure offers guidance for designing therapeutic interventions against the SARS-CoV-2 infection.


■ INTRODUCTION
SARS-CoV-2, a positive-sense single-stranded RNA virus from the β-coronavirus family, 1 is the causative agent of the COVID-19 pandemic that killed millions of people and brought the world economy to a grinding halt. 2,3 The SARS-CoV-2 genome encodes four structural proteins: spike (S) glycoprotein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. 4,5 All play crucial roles in the viral life cycle and pathogenicity, including host immunity evasion. 6 Due to its important role in genome packaging and ribonucleoprotein (RNP) formation, the N protein represents a potential target for therapeutic interventions. 7−9 The N protein comprises two folded domains, the Nterminal (N NTD , residues 40−174) and C-terminal (N CTD , residues 246−365) domains, connected by an ∼70 amino acid linker region that contains a 13-residue serine/arginine motif, as well as extensive intrinsically disordered regions (IDRs) at the N-and C-termini 10−15 (Figure 1a). All domains including the N NTD play an important role in the RNA genome interaction. 16−18 Several structures of β-coronavirus N NTD domains have been reported, all of which possess the same architecture, resembling a right hand. 19−24 The core structure is made of a four-stranded antiparallel β-sheet, the palm, from which the β2, β3 hairpin prominently protrudes. It contains several basic residues, and this basic finger and the palm have been implicated in RNA binding. 25 The loop connecting β2 and β3 is flexible, in agreement with the missing density in this region of most X-ray structures (see below). 19,26 The N-terminal disordered tail projects outward and may contribute to RNA binding. 19 Here, we report the atomic-resolution structure of crystalline N NTD , determined by combining X-ray crystallography and solid-state magic angle spinning (MAS) NMR spectroscopy. The protein crystallized in the P2 1 2 1 2 1 space group with four chains in the asymmetric unit, and the X-ray structure was solved at a 1.7 Å resolution. The MAS NMR structure of an individual N NTD chain, at 0.7 Å rmsd resolution, was determined using a single crystalline U− 13 C, 15 N−N NTD sample, based on 2968 nonredundant 13 C− 13 C, 15 N− 13 C, and 15 N− 1 H distance restraints. Several inter-chain contacts were identified in 13 C− 13 C correlation experiments, both for chains in the asymmetric unit as well as across asymmetric units. Side-chain proton chemical shifts were assigned from high-frequency (100 kHz) MAS NMR correlation experiments and provided important structural information, such as the tautomeric state of the H107 residue. Our results illustrate the power of integrating orthogonal structural techniques, here MAS NMR and X-ray diffraction, for assessing details of protein conformations. The atomic-resolution structure of crystalline N NTD reported here will guide the development of small-molecule inhibitors and biologics for treatment as well as biosensors for the detection of SARS-CoV-2 infection. 27 (d) Sequential assignments for the D44−G47 stretch of residues are illustrated with representative strips of 2D NCACX (gray), NCOCX (blue), 1 H-detected three-dimensional (3D) (H)CANH (black), and (H)CONH (pale blue) spectra. (e) Selected strips from the 1 Hdetected 3D (H)CCH spectrum for residues T77 and P42 illustrating 13 C and 1 H side-chain resonance assignments. CORD, NCACX, and NCOCX spectra were recorded at an MAS frequency of 14 kHz; (H)CANH and (H)CONH spectra were acquired at an MAS frequency of 60 kHz; and HETCOR and (H)CCH spectra were acquired at an MAS frequency of 100 kHz. The number of scans and the number points in the direct and indirect dimensions are as follows: 2D (H)NH HETCOR -32 scans, 1024 t 2 points, 1034 t 1 points; 2D (H)CH HETCOR -64 scans, 1024 t 2 points, 2310 t 1 points; 2D CORD -192 scans, 2048 t 2 points, 840 t 1 points; 2D NCACX -2048 scans, 2048 t 2 points, 96 t 1 points; 2D NCOCX -1536 scans, 3072 t 2 points, 96 t 1 points; 3D (H)CANH -48 scans, 2048 t 3 points, 112( 15 N) t 2 points, 32( 13 C) t 1 points; 3D (H)CONH -32 scans, 2048 t 3 points, 112( 15 N) t 2 points, 32( 13 C) t 1 points; and 3D (H)CCH -8 scans, 1024 t 3 points, 264 ( 13 C) t 2 points, 264 ( 13 C) t 1 points.

■ RESULTS
Resonance Assignments. Chemical shift assignments and distance restraints for N NTD structure calculation were obtained using a single sample of fully protonated crystalline U− 13 C, 15 N−N NTD comprising residues 40−174 (current construct residues 2−136; Figure 1a; see the Materials and Methods section for experimental details). A total of eleven 2D and three 3D 1 H-and 13 C-detected high-frequency (100 and 60 kHz) MAS NMR experiments were recorded (Figure 1b−e and Supporting Information Table S1). The spectra are of remarkably high resolution, with line widths as narrow as 35 Hz for 15 N, 48 Hz for 13 C, and 174 Hz for 1 H N (Figure 1b).
2D CORD, 28 NCACX, and NCOCX at a 25 ms mixing time, as well as 1 H-detected 2D (H)NH HETCOR, 3D (H)CANH, and (H)CONH spectra (Figure 1c,d), were used for sequential backbone assignments, and 13 C and 15 N chemical shifts are complete for 128 of 136 residues. For five residues, F28, P84, P113, P124, and E136, partial backbone chemical shift assignments were obtained, and, for 119 residues, backbone amide proton (H N ) chemical shifts were assigned. The resonances of the first two residues, R2 and R3, are missing in the spectra, likely due to disorder. Overall, good agreement is observed between 1 H and 15 N chemical shifts determined in this work and those reported previously from solution NMR. 25,29 MAS NMR assignments for a representative stretch of residues D44−G47 are illustrated in Figure 1d.
Side-chain 13 C chemical shifts and inter-residue correlations were obtained from 2D NCACX, NCOCX, and CORD spectra, the latter acquired with 25, 100, and 500 ms mixing times (Figures 1c and 2a). High spectral resolution permitted the unambiguous assignment of numerous cross-peaks, including those corresponding to aliphatic-to-aromatic (left panel) and aliphatic-to-aliphatic (right panel) side-chain correlations ( Figure 2a). To determine side-chain and backbone 1 H chemical shifts, a 3D (H)CCH correlation experiment was recorded at an MAS frequency of 100 kHz ( Figure 1e). In conjunction with spectra acquired at an MAS frequency of 60 kHz, 84 side-chain proton resonances for 71 residues and H α resonances for 65 residues were assigned. For 11 Ala, 3 Val, 4 Ser, and 1 His residues, complete 13 C, 15 N, and 1 H backbone and side-chain chemical shifts were obtained. Overall, assignments for 132 residues were attained ( Figure  S1) on the basis of 3728 cross-peaks in various spectra ( Table  1). All chemical shifts are summarized in Table S2 of the  Supporting Information. Gratifyingly, many side-chain protons of aromatic residues could be unambiguously assigned from the 1 H-detected 100 kHz MAS NMR spectra (Figure 1b). For example, for W70 and W94, located in the β-sheet core and assumed to be involved in RNA binding, side-chain protons were assigned fully (W70) or partially (W94). Moreover, the tautomeric state of H107 was determined (see below).
Structure of a Single N NTD Chain Determined by MAS NMR. The structure of an N NTD single chain was calculated using 2968 nonredundant distance and 101 ϕ/ψ torsion angle restraints. Of these, 2197 are unambiguous 13 C− 13 C, 763 are  Table 2. With nearly 22 restraints/residue on average, the N NTD structure determined in this study represents a notable technical advance being one of only two MAS NMR structures of proteins larger than 100 residues per chain determined using more than 20 restraints/ residue and reaching the maximum accuracy and precision attained for MAS NMR protein structures 30 (see below).
X-ray Crystal Structure of the N NTD . The protein crystallized in the P2 1 2 1 2 1 orthorhombic space group with four monomers (chains A−D) in the asymmetric unit (Figures 3a and S4a of the Supporting Information). Two views of the four chains are provided in Figure 3a, and chain A is depicted in Figure 3b. Details of the β2, β3 hairpin and loop region, as well as the difference electron density map, are shown in Figure 3c. Complete statistics for X-ray data collection, phasing, and refinement are provided in Table 3. The average pairwise rmsd value between the four chains in the asymmetric unit is 0.5 ± 0.1 Å for the backbone atoms (excluding common missing residues in all four chains, R2−N9, Q20−D25, R57− P68, and P124−E136) ( Figure S5b and Table S3 of the Supporting Information). A positively charged region, comprising arginine residues in the β-sheet (R50, R51, R54, R55, R69) and at the tip of the β-hairpin finger (R57, K62), may possibly contribute to RNA binding ( Figure S4c of the Supporting Information). 19,25 Interesting details about the intratetramer interfaces in the crystal can be noted in the structure, with five unique types of contacts formed by the residues within the tetramer ( Figure  4a). Specifically, (i) the A−B interface comprises several residues (T18, H21, I36, R54, R56−G59, K64, L66, S67, V120, Q122, Y134, and A135) from both chain A and chain B; (ii) the A−C interface is very small and involves residues G59 and D60 of chain A contacting K131 of chain C; (iii) the B−C interface comprises several residues, such as R30−Q32, P42, D43, E98−L101, and P124−T127 of the chain B palm region, which are in contact with the C-terminal tail (residues L121− L129, K131, G132, Y134, A135), as well as H21, G22, and K23 of chain C; (iv) the B−D interface packs the palm regions of chains B and D against each other; and (v) the D−C interface comprises residues I56 and P113−A117 of chain D and T16, H21, A117, A118, V120, and Q122 of chain C.
The X-ray and the MAS NMR structures of the individual chains are in good agreement, with a backbone rmsd of 1.1 Å between the X-ray structure (averaged over the four chains in the asymmetric unit) and the MAS NMR structure (averaged over the ensemble of 10 lowest-energy structures) ( Figure S5 and Table S3 of the Supporting Information). Upon exclusion of chain D, which possesses the highest degree of disorder in the X-ray structure, the corresponding value becomes 0.7 Å. The average pairwise backbone rmsd between the four chains in the X-ray structure and within the ensemble of 10 lowestenergy MAS NMR structures are, both, 0.5 ± 0.1 Å ( Journal of the American Chemical Society pubs.acs.org/JACS Article D of the Supporting Information). Side-chain conformations for most residues in all four chains of the X-ray structure vary little, except for residues N10, R30, R55, and I56, which exhibit major differences ( Figure S6 of the Supporting Information). Unlike the MAS NMR structure, which was determined using distance restraints or/and chemical shifts for most residues, except R2, R3, and E136, density is either missing or very weak for residues R2−N9 and E136 in all chains and for residues Q20−D25, R57−P68 and P124−E136 in chain D.
These unique intra-and intertetramer interfaces are reflected in distinct correlations in the MAS NMR spectra. In the (H)NH spectrum recorded at an MAS frequency of 100 kHz, multiple resolved peaks or broad (unresolved) peaks are observed for residues that are found in several different local environments, with 15 N peak widths of ∼85−110 Hz ( Figure  5b), whereas those for amino acids in single conformations are ∼40−60 Hz (Figure 5a,c). Examples of correlations corresponding to intratetramers A−B, A−C, and B−D interfaces, as well as to the intertetramer interfaces, are shown in Figure  4a,b, bottom panels.
One notable example of unique intratetramer contacts, evidenced by multiple cross-peaks in the MAS NMR spectra, is seen for the H21−Y134 pair of residues (Figure 4a, bottom panel). It is evident from the X-ray structure that the interchain distances are much shorter than the intrachain ones (3.5−4.6 and 7.0−8.0 Å, respectively); hence, only interchain correlations are expected in the spectra ( Figure 6). Another interesting example involves residues D60 and M63 in the β2,  Figure  7). In addition to the intra-and intertetramer contacts discussed above, we also observed multiple conformers for many residues, as is evident from their distinct backbone and sidechain chemical shifts. For example, T77 exhibits several resolved resonances with unique chemical shifts each ( Figure  4c, left panel). A unique T77C β −A117C′ cross-peak is found for one of the two conformers (designated as conformer b, T77 b ) in the 100 ms mixing time CORD spectrum. This correlation is missing for the second conformer, T77 a . Both conformers exhibit significant chemical shift differences (ΔN = 1.4 ppm, ΔC β = 0.3 ppm, ΔC γ = 0.3 ppm, ΔC′ = 0.6 ppm), consistent with distinct local environments for T77. Indeed, in the X-ray structure, T77 in 0 A and 0 B has no intra-or intertetramer contacts within 7 Å, consistent with this conformer being T77 a . In contrast, T77 in chains 0 C and 0 D exhibits intertetramer contacts with A117 from chains 4 B and 3 A , respectively, therefore suggesting that these resonances correspond to the T77 b conformer (Figure 4c, right panels). Likewise, at least two distinct conformations are seen for A52, whose backbone chemical shifts are different (ΔN = 2.0 ppm, ΔC α = 0.4 ppm), suggesting that they exist in unique local environments ( Figure S7, top panel, Supporting Information). This finding is fully consistent with the X-ray structure ( Figure  S7, bottom panel, Supporting Information), where A52 in 0 A and 0 B forms intertetramer contacts with D43 and L101 from chains 6 C and 5 D , respectively, while A52 in chains 0 C and 0 D forms contacts with E24 and K27 from chains 8 A and 0 B , respectively.
Tautomeric State of Histidine-107 in the Crystal. In the 1 H-detected 2D (H)NH spectrum acquired with a CP contact time of 0.3 ms, H107 gives rise to two distinct 15 (Figures 1b and 8a). In 2D (H)CH spectra, multiple C ε1 −H ε1 and C δ2 −H δ2 correlations are also observed (Figure 8a). Taken together, these results suggest the presence of local heterogeneity around H107, consistent with two distinct local environments seen in the X-ray crystal structure (Table S4 of 35,36 The deshielded H ε2 resonance at δ( 1 H) ∼ 12.5 ppm suggests that the H107 imidazole may be involved in hydrogen bonding or close to a negatively charged group. 34,37 The presence of a 15 N ε2 −H ε2 correlation in the (H)NH spectrum acquired with a 0.3 ms CP contact time also suggests a short nitrogen− hydrogen distance, possibly a directly bonded N ε2 −H. Indeed, in the X-ray crystal structure, H107 is in close proximity to D44 and the interatomic H107N ε2 −D44O δ1,δ2 distances are 2.9, 2.9, 3.2, and 3.4 Å in chains A, B, C, and D, respectively (Figure 8b). Taken together, these data indicate that the (H107)N ε2 −H is close to the carboxylate side chain of D44.

■ DISCUSSION
The NTD domain of the SARS-CoV-2 virus N protein has been previously structurally characterized by X-ray crystallography and solution NMR. 16,19,25 Here, we present a structure that was determined by integrating MAS NMR and X-ray diffraction, providing important novel findings of distinct conformers, made possible by the remarkably high resolution of the MAS NMR spectra. The structural heterogeneity of N NTD is an outcome of crystallization, as seen in other N NTD crystal structures, 19,26,31 and underscores the ability of the protein to form multiple types of contacts involving distinct conformers with unique local environments.
From the technical standpoint, the current study represents a notable advance for determining protein structures by MAS   Notable is the complementarity of information obtained by MAS NMR and X-ray diffraction. In the X-ray structure, the atomic level details for individual chains and information on the quaternary arrangement in the crystal are obtained, while the strength of MAS NMR lies in providing information about dynamically disordered regions, proton positions, protonation and tautomeric states, and contacts with water molecules. To understand the dynamics of the different regions of N NTD in solution and in the crystalline forms and their role in RNA binding, it will be interesting in future studies to perform measurements of relaxation rates, chemical shifts, and dipolar anisotropy tensors.

■ CONCLUSIONS
We have determined the structure of SARS-CoV-2 N NTD by integrating MAS NMR and X-ray diffraction. Our combined approach provided atomic details of packing interfaces as well as information about disordered residues at the N-and Ctermini and the functionally important RNA binding, β-hairpin loop. In addition, 1 H-detected experiments at an MAS frequency of 100 kHz permitted the assignment of side-chain proton chemical shifts not available by other means. The present structure offers guidance for designing therapeutic interventions against the SARS-CoV-2 infection.   Cells were opened after treatment with 1 mM phenylmethylsulfonyl fluoride (PMSF) by sonication at 40% power for 5 min (15 s pulse on and 45 s pulse off) on ice. The cellular lysate was clarified by centrifugation at 14,000g for 1 h at 4°C. His 6 -tagged N NTD was purified by affinity chromatography over a 5 mL HisTrap HP column (GE Healthcare). For elution, a gradient of 20−500 mM imidazole in 20 mM tris−HCl (pH 8.0), 500 mM NaCl, and 0.02% NaN 3 was employed. The His 6 -tag was cleaved with TEV protease (1:25 ratio of TEV protease to His 6 −U− 13 C, 15 N−N NTD ) for 12−16 h at 4°C and again fractionated over a 5 mL HisTrap HP column (GE Healthcare). Fractions were eluted in 20 mM tris−HCl, 500 mM NaCl, 20 mM imidazole, 0.02% NaN 3 , pH 8.0, and the pure protein was bufferexchanged into crystallization buffer (20 mM tris−HCl, 50 mM NaCl, pH 6.0) and NMR buffer (20 mM tris−HCl, 150 mM NaCl, 90:10 H 2 O/D 2 O, pH 8.0). The buffer-exchanged N NTD was concentrated to 12 mg/mL for solution NMR and 30 mg/mL for crystallization.
Crystallization of N NTD . Small-scale crystallization was carried out at room temperature (∼20°C) using the sitting-drop vapor diffusion. Two microliters of U− 13 C, 15 N−N NTD (20 mM tris−HCl, 50 mM NaCl, pH 6.0) was mixed with 2 μL of crystallization buffer (100 mM MES, 30% PEG4000, pH 6.5), modified from a previously published crystallization condition (PDB: 6WKP). 26 U− 13 C, 15 N−N NTD for MAS NMR experiments was crystallized using a large-scale sitting-drop method based on the volumetric proportions of 500 μL sitting-drop crystallization wells ( Figure S9 of the Supporting Information). A series of presterilized Petri dishes were used to form a concentric sitting-drop vessel with a reservoir (volume capacity of 25−75 mL) and three droplet wells (optimal volume of 300−1000 μL). Similar to the small-scale crystallization, the crystallization droplet mixture comprised 250 μL of N NTD and 250 μL of crystallization buffer. The large-scale sitting-drop vessel was sealed using vacuum grease and left undisturbed at 20°C for 5 days. Once crystallization was complete, the protein crystals were harvested and packed into a Bruker 1.3 mm rotor by ultracentrifugation at 10,000g for 15 min at 4°C. The fully packed 1.3 mm rotor contained 3.6 mg of hydrated protein crystals. The same sample (∼0.5 mg) was subsequently packed in a 0.7 mm rotor for experiments at 100 kHz MAS and 20.0 T.
Diffraction Data Collection and Structure Determination. Xray diffraction data of protein crystals were collected at beamline 12-2 at the Stanford Synchrotron Radiation Lightsource (SRRL). All diffraction data used for analysis were collected from crystals grown in 100 mM MES, 30% PEG4000, pH 6.0 at 100 K. All diffraction data were indexed and integrated using the program XDS 38 and scaled using the program AIMLESS from the CCP4 suite. 39 The structure was solved by molecular replacement (MOLREP, CCP4 suite) using one monomer of PDB:ID 6M3M. Structure refinement was carried out in Phenix 40 with manual building in COOT 41 13 C CP rf field strength was set to 30 kHz; for forward and back CP, linear amplitude ramps on 1 H were 80−100 and 100−80%; the 1 H rf field strengths were set at 138 and 132 kHz; the contact times were 600 and 175 μs, respectively. The CC RFDR mixing time was 0.56 ms. For all spectra, the 1 H rf field strengths for water suppression and proton decoupling were set at 1/4 ω r , and a WALTZ sequence at 10 kHz was used for heteronuclear decoupling of both 13 C and 15 N. An additional 2D (H)NH spectrum was recorded at 60 kHz MAS, using a 1.3 mm HCN probe. The CP contact time was 4 ms, and the remainder of all conditions were identical to those at 14.1 T (see above).
Data Processing. All MAS NMR data were processed using Bruker TopSpin and NMRPipe. 47 1 H resonances are referenced with respect to water at 4.7 ppm and 13 C and 15 N to the external standards adamantane and ammonium chloride, respectively. All 2D and 3D data sets were processed by applying 30, 45, 60, and 90°shifted sine bell apodization, followed by a Lorentzian-to-Gaussian transformation in all dimensions. Forward linear prediction to twice the number of original data points was applied in the indirect dimension for some data sets, followed by zero filling. 2D and 3D 1 H-detected data sets were processed with Gaussian and/or square sine window apodization and quadrature baseline correction.
Structure Calculation of SARS-Cov2 N NTD . The MAS NMR structure of a single N NTD chain was calculated in Xplor-NIH version 2.53 51−53 using 13 C− 13 C, 15 N− 13 C, and 1 H− 15 N distance restraints, extracted from 2D CORD (100, 250, and 500 ms mixing times), NCACX, NCOCX, and (H)NH HETCOR spectra and backbone dihedral angles predicted by TALOS−N 54 from the experimental 1 H, 13 C, and 15 N chemical shifts. The bounds for the distance restraints were set to 1.5−6.5 Å (4.0 ± 2.5 Å) and 2.0−7.2 Å (4.6 ± 2.6 Å) for intra-and inter-residue restraints, respectively, consistent with our previous studies. 30,55 Calculations were seeded using the primary sequence as extended strands. One thousand structures were generated with molecular dynamics simulated annealing in torsion angle space with two successive annealing schedules and a final gradient minimization in Cartesian space, essentially as described previously 30,55 and detailed below.
Two successive annealing schedules were used, the first in vacuum with the REPEL module and the second with an implicit solvent refinement using the EEFx module. 56 The 10 lowest-energy structures were selected and served as input for the second schedule, and the 10 lowest-energy structures were selected and served as input for the final ensemble (PDB: 7SD4). Standard terms for bond lengths, bond angles, and impropers were applied to enforce the correct covalent geometry.
The first annealing calculation was essentially identical to that reported previously, 30,55 with initial random velocities at 3500 K constant-temperature molecular dynamics run for the shorter of 800 ps or 8000 steps, with the time step size allowed to float to maintain constant energy. Subsequently, simulated annealing calculations at reduced temperatures in steps of 25−100 K were carried out for the shorter of 0.4 ps or 200 steps. Force constants for distance restraints were ramped from 10 to 50 kcal/(mol·Å) 2 . Dihedral angle restraints were disabled for high-temperature dynamics at 3500 K and subsequently applied with a force constant of 200 kcal/(mol·rad 2 ). The force constant for the radius of gyration was geometrically scaled from 0.002 to 1, and a hydrogen bond term, HBPot, was used to improve hydrogen bond geometries. 57 After simulated annealing, structures were minimized using a Powell energy minimization scheme.
For the second schedule performed in implicit solvent, all parameters were set as in the example EEFx of Xplor-NIH. Annealing was performed at 3500 K for 15 ps or 15,000 steps, whichever was completed first. The starting time step was 1 fs and was self-adjusted in subsequent steps to ensure conservation of energy. Random initial velocities were assigned about a Maxwell distribution at the starting temperature of 3500 K. Subsequently, the temperatures were reduced to 25 K in steps of 12.5 K. At each temperature, 0.4 ps dynamics were run with an initial time step of 1 fs. Force constants for distance restraints were ramped from 2 to 30 kcal/(mol·Å 2 ). The dihedral restraint force constants were set to 10 kcal/(mol·rad 2 ) for hightemperature dynamics at 3,000 K and 200 kcal/(mol·rad 2 ) during cooling. After the EEFx module, structures were minimized using a Powell energy minimization scheme.
Structure Analysis and Visualization. Atomic rmsd values were calculated using routines in Xplor-NIH (version 2.53). 51−53 The visualization of structural ensembles was rendered in PyMOL, 58 using in-house shell/bash scripts. Secondary structure elements were classified according to STRIDE 59 and manual inspection. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c03320.
Summary of resonance assignment of SARS-CoV-2 N NTD ; X-ray crystal structure; 10 lowest-energy conformers of MAS NMR structures; all and selected sidechain conformations of MAS NMR and X-ray structures; multiple resonances for A52 in the MAS NMR spectra; histidine protonation state in solution NMR; mass spectrum, crystallization, and purification of N NTD ; 2D 1 H− 15 N HSQC NMR spectrum of U− 15 N−N NTD ; summary of all NMR experiments; MAS NMR and solution NMR chemical shifts of N NTD ; rmsd values between X-ray and MAS NMR structures; intra-and intertetramer contacts in N NTD crystals; and author contributions (PDF)