DARR spectrum of U-¹³C,¹⁵N HIV-1 CA conical assemblies at 21.1 T. Aliphatic region (right) and carbonyl region (left). The 1D projections in the ω₂ and ω₁ dimensions corresponding to the resolved cross-peaks for Thr residues are shown and illustrate the remarkable spectral sensitivity and resolution. The spectrum was acquired as (3072 × 216) (complex × real) matrices with 220 scans; the recycle delay was 2 s. The spectral widths were 100 kHz and 50 kHz in the ω₂ and ω₁ dimensions, respectively, and the DARR mixing time was 50 ms. Cross-peaks corresponding to the different amino acid types appear in distinct, resolved regions of the spectrum and are color coded as follows: Ala, red (C^α-C^β and C^β-C' correlations); Gly, orange (C^α-C' correlations); Ile, yellow (C^α-C^δ1, C^β-C^γ1, C^β-C^γ2, and C^γ1-C^δ1 correlations); Leu, grey (C^β-C^γ correlations); Pro, purple (C^α-C^γ correlations); Ser, light green (C^α-C^β correlations); Thr, blue (C^α-C^β and C^β-C^γ correlations); Val, bright green (C^α-C^β correlations).

Superposition of DARR spectra recorded on spherical (black) and conical (red) assemblies of U-¹³C,¹⁵N CA at 14.1 T. The spectra of the spherical assemblies were acquired as (1000 × 400) (complex × real) matrices using 80 scans and a recycle delay of 2 s, a spectral width of 50 kHz in both dimensions and the DARR mixing time of 10 ms. The acquisition parameters for the spectra of the conical assemblies are given in the caption to Figure 3.

Comparison between solid-state and solution secondary chemical shifts for CA conical assemblies. C^α shifts that reside in α-helical regions of the protein exhibit positive secondary shifts, while those located in the β-strands and other secondary structure elements are negative.

Assemblies of HIV-1 CA exhibiting conical, spherical, and tubular morphologies. (a), Conical assembly. From left to right: a negatively stained TEM image, confocal image (fluorescence mode), cryo-SEM image, confocal image (fluorescence mode) of the sample after magic angle spinning. (b), Spherical assembly. From left to right: confocal image (fluorescence mode) of the solution containing the assemblies, cryo-SEM image, confocal image (fluorescence mode) of the dried out sample, confocal image (fluorescence mode) of the sample after magic angle spinning. (c), Tubular assembly. From left to right: negatively stained TEM image, confocal image (fluorescence mode), confocal image (fluorescence mode) of the dried out sample, confocal image (fluorescence mode) of the sample after magic angle spinning.

14.1 T DARR spectra of conical assemblies acquired on a set of uniformly and specifically labeled CA. (a) The DARR spectrum of U-¹³C,¹⁵N sample; (b) the superimposed DARR spectra of U-¹³C,¹⁵N sample (black) and U-¹³C,¹⁵N-Ala/U-¹³C,¹⁵N-Thr sample (red), and (c) U-¹³C,¹⁵N-His/U-¹³C,¹⁵N-Tyr sample (expansions of the aliphatic (right) and carbonyl (left) regions illustrate the one-bond correlations for His and Tyr residues). The spectra in (a) were collected as (2000 × 400) (complex × real) matrices with 200 scans, a recycle delay of 2 s, and a spectral width of 50 kHz in both dimensions. The spectra in (b) were obtained as (2000 × 512) (complex × real) matrices with 256 scans, a recycle delay of 1 s, and spectral widths of 50 kHz and 25 kHz in the ω₂ and ω₁ dimensions, respectively. The spectra in (c) were acquired as (1000 × 128) (complex × real) matrices with 640 scans, and a recycle delay of 1.5 s and spectral widths of 50 kHz and 33.3 kHz in the ω₂ and ω₁ dimensions, respectively. The DARR mixing time was 10 ms for all data sets.

Selected 2D planes of the 3D NCACX spectrum of U-¹³C,¹⁵N CA conical assemblies. Spectral resolution in planes a–g and j is high enough to allow assignment of spin systems, while planes h and i exhibit substantial resonance overlap. The spectrum was acquired as (1000 × 40 × 32) (complex × real × real) matrices with 256 scans and a recycle delay of 2 s. The spectral widths were 50 kHz, 4.167 kHz, and 8.333 kHz in the ω₃, ω₂ and ω₁ dimensions, respectively. SPECIFIC-CP (τ_mix = 6.2 ms) was employed for the first NCA transfer followed by the DARR mixing (τ_mix = 20 ms) resulting in predominantly one- and two- bond correlations. (k) Sequential backbone connectivity for the sequence stretch A105–L111 in conical assemblies based on 3D NCOCX, NCACX, and NCACB experiments at 14.1 T. The residue names are shown on top of the spectra at their ¹⁵N chemical shift plane. Negative cross-peaks resulting from two-bond N-C^β correlations in the NCACB spectra are displayed in red. The NCOCX spectrum was acquired as (1000 × 36 × 40) (complex × real × real) matrices with 280 scans and a recycle delay of 2 s. The spectral widths were 50 kHz, 4.167 kHz, and 6.250 kHz in the ω₃, ω₂ and ω₁ dimensions, respectively. SPECIFIC-CP (τ_mix = 6.2 ms) was employed for the first NCO transfer followed by the DARR mixing (τ_mix = 15 ms) resulting in predominantly one- and two-bond correlations. The NCACB spectrum was acquired as (1000 × 32 × 32) (complex × real × real) matrices with 400 scans and a recycle delay of 2 s. The spectral widths were 50 kHz, 4.167 kHz, and 8.333 kHz in the ω₃, ω₂ and ω₁ dimensions, respectively. SPECIFIC-CP (τ_mix = 6.2 ms) was employed for the first NCA transfer followed by the DREAM mixing (τ_mix = 3 ms) to establish the CA-CB correlations.