CA pentamer model building

The initial coordinates of the cross-linked pentamer, P17C/T19C and N21C/A22C, model were directly derived from the crystal structure (PDB accession code 3P05), missing loops were added using Modeler [37]. Subsequently, all cysteine residues were mutated back to the wild-type HIV-1_NL4-3 wild-type sequence, and the complete pentamer was then rigid body docked, using Chimera, into the electron microscopy density of the pentamer obtained from intact particles (EMD accession code EMD-3466). To obtain an atomistic model of the 5MCY pentamer, flexible fitting was performed employing the MDFF method as follows [38]. Ions near the surface of the protein were placed using Cionize [39]. Bulk water and Na/Cl ions were then added using VMD, setting the total concentration of NaCl to 150 mM. Each of the resulting systems contained 100,000 total atoms, including solvent. The systems were equilibrated at 310 K and 1 atm for 5 ns while applying positional restraints on all heavy atoms of the protein. MDFF was then applied for 10 ns with a grid scaling of φ = 0.05 coupled only to backbone heavy atoms; EMD-3466 was used as the target density. Domain restraints were applied to maintain the structural integrity of each individual CA domain in the pentamer. Additional restraints were applied in the form of extra bonds to maintain secondary structure integrity and to prevent transitions of cis/trans bonds and chirality errors [40]. MDFF was performed using NAMD 2.12 [41] with the CHARMM36m force field. Simulated density maps needed for determining cross correlation between model and EM data were generated using VMD [41]. The resulting model was labeled 5MCY, as its backbone resembles the PDB entry 5MCY.

CA hexamer model building

The initial coordinates of the HIV-1 CA hexamer (S12A and S12B Fig) were generated by applying a 6-fold symmetry operation onto a native full-length HIV-1 CA (PDB accession code 4XFX). The 2 loops between residues 5 to 9 and residues 222 to 231, missing in the original structure, were built using Modeler [30]. Once the hexamer was built, the protonation states of titratable residues, namely histidine, asparagine, lysine, and cysteine, were assigned using PDB2PQR [31] (pH 7.4).

Molecular mechanics parameterization of NTPs, IP₆, and BHC

Parameters for the negatively charged small molecules (S13 Fig), except ATP, which has parameters available in the CHARMM general force field, were derived by analogy following the CGENFF protocol [42]. The parameter penalties and charge penalties in each generated parameter files were less than 10 indicating good analogy with the available atom types present in CGENFF. A magnesium ion was added to the triphosphate group present in dNTPs. The coordination number of the magnesium ion with the phosphate group of the dNTP was constrained using coordnum in Colvars [43].

Simulation setup

Small charged molecules such as NTP/IP₆/BHC were placed in the central cavity of the CA hexamer model. The models were then solvated using the TIP3P water model [44]. Additionally, excess of TIP3P water molecules were deleted to transform the cubic water box into a hexagonal orthorhombic cell of dimension 92.328 Å in the plane and 90 Å in the ẑ direction. The length of the system in the ẑ direction provided sufficient solvent padding, greater than 24 Å, to avoid interactions between periodic images. Na and Cl ions were then added to neutralize the system using the CIONIZE plugin in VMD [45], and the bulk salt concentration was set to the physiological concentration of 150 mM, as previously described by the authors [46]. The total number of atoms of the resulting CA hexamer models was 60,000 (S12C and S12D Fig). Solvated WT and K25A, K25N, K25E, and K25E/E28K CAs were derived using the mutator plugin in VMD.

System minimization and equilibration

The solvated systems were then subjected to minimization in 2 stages, both using the conjugated gradient algorithm with linear searching as implemented in NAMD [39]. Each stage consisted of 10,000 steps of energy minimization. During the first stage, only water molecules and ions were free to move, while the CA protein and NTP/IP₆/BHC molecules were fixed. In the second stage, the backbone atoms of the CA protein were restrained with a force constant of 10.0 kcal mol⁻¹ Å⁻². Convergence of the minimization procedure was confirmed once the variance of the gradient was below 0.1 kcal mol⁻¹ Å⁻¹. Following minimization, the systems were tempered from 50 K to 310 K in increments of 20 K over 1 ns. Subsequently, the systems were equilibrated at 310 K for 100,000 steps, while the protein backbone atoms were restrained. Equilibration of the systems was assessed using the RMSD trace versus time plateauing at 2 Å (S14A Fig), the local RMSF per amino acid showing fluctuations less than 1 Å for residues located in helical regions (S14B Fig), and by monitoring the convergence of the radius of the pore in the CA hexamer (S14C Fig). Then positional restraints were gradually released at a rate of 1.0 Kcal mol⁻¹ Å⁻² per 400 ps from 10.0 Kcal mol⁻¹ Å⁻² to 0.0 Kcal mol⁻¹ Å⁻². All MD simulations were performed using NAMD 2.12 with the CHARMM36m force field [39,47,48]. An internal time step of 2 fs was employed in the multistep r-RESPA integrator as implemented in NAMD, bonded interactions were evaluated every 2 fs, and electrostatics were updated every 4 fs. Temperature was held constant at 310 K using a Langevin thermostat with a coupling constant of 0.1 ps⁻¹. Pressure was controlled at 1 bar using a Nose-Hoover Langevin piston barostat with period and decay of 40 ps and 10 ps, respectively. The Shake algorithm was employed to constraint vibrations of all hydrogen atoms. Long-range electrostatics was calculated using the particle-mesh-Ewald summation with a grid size of 1 Å and a cutoff for short-range electrostatics interactions of 12 Å.

Gibbs free energy calculations

Progress variables (PVs), akin to reaction coordinates, for all free-energy molecular dynamics calculations were chosen as the location on the z axis of the center of mass of the small-molecule(s) of interest. The origin of the progress variable was set to the center of mass of C_α atoms in the N-terminal domain of the CA hexamer (Fig 1C). One- and two-dimensional (2D) potentials of mean force (PMFs) along the PVs were calculated using the Hamiltonian Replica-exchange/Umbrella Sampling (HREX/US) method [43,49,50] implemented in NAMD 2.12 [39,43,51]. The initial coordinates for the HREX/US windows were derived from constant-velocity steered MD (SMD) simulations in which molecules were pulled along the PV at a rate of 0.1 nm/ns. The center of mass of the small-molecules were positionally resta rained in the US windows with a harmonic force constant of 2.5 Kcal mol⁻¹ Å⁻² using Colvars [49]. The width of all US windows was set to 0.75 Å; except for single nucleotide HREX simulations (simulation #1 to 8) in which the window width was set to 1.0 Å. The number of US windows in the present HREX simulations were chosen so that the nucleotide translocation from capsid exterior to interior through the central cavity was uniformly sampled (Fig 1C).

2D HREX/US simulations were employed to study the cooperativity between small molecules for translocation. For this purpose, 2 PVs were employed: PV1 that determined the location of dATP, and PV2 that determined the location of dATP, IP₆, or BHC in the CA cavity. The initial configurations for the 2D simulations were derived by pulling dATP using constant velocity steered MD [52] at 0.1 nm/ns along PV1, while the center of mass of dATP/IP₆/BHC was restrained with a harmonic force constant of 2.5 Kcal mol⁻¹ Å⁻² using Colvars (cycle 1, S15A–S15C Fig). A seeding method similar to that reported [53] was employed to generate new simulation windows along PV2 as follows. For each conformation resulting from the SMD simulation, 2 new seeds were generated by displacing by ±0.75 Å the harmonic restraints for dATP/IP₆/BHC along PV2 (cycle 2, S15A–S15C Fig). Subsequently, 5 ns of HREX/US MD simulation were performed. The last configuration from each replica was then used as the initial seed for a subsequent cycle where the harmonic restraints were displaced by another ±0.75 Å along PV2 (cycle 3, S15A–S15C Fig). The seeding process was repeated one last time and production 2D HREX/US simulations (production runs, S15A–S15C Fig) were performed for 30 ns per window. During each HREX/US simulation, exchange of the harmonic potential between neighboring replicas was attempted every 1000 steps (2 ps). Exchanges were attempted randomly between successive replicas; trial attempts for all replicas were equally probable. The following Metropolis Monte Carlo exchange criterion [50] was employed where T = 310 K, k_B is the Boltzmann constant, q_i and q_j denote the 3D conformations for 2 replicas i and j, and U_i and U_j represent the potential energies derived from the restrained Hamiltonian evaluated at the indicated conformation.

Potentials of mean force were derived from the resulting sampling in each of the US windows using the weighed histogram analysis method (WHAM) [53,54]. In WHAM, PMF bins are obtained from US/HREX simulation windows. Convergence of the 1D US/HREX calculations was characterized by the changes in the resulting PMF in trajectory increments of 10 ns (S16 Fig). That is, simulations were considered to have converged once the maximum change in one of the PMF bins resulting from adding more simulation data was less than 1 Kcal mol⁻¹.

Convergence of the 2D calculations was examined by means of the fluctuations of the average root mean squared error for each PMF bin (RMSE) (S17A Fig). Each 2D HREX/US simulation was divided into 60 time-slices of width 0.5 ns. Then, the PMF was calculated for each slice using WHAM. Using the last slice as the reference, the RMSE of the k-th slice was calculated using the equation: where the sum runs over the total number of PMF bins (N_bins), G_{i, k} and G_{i, reference} are the free energies at the i-th PMF bin for the k-th and last slice, respectively. Convergence of 2D PMF was established once the RMSE was not larger than 3.0 kcal/mol. The bins before convergence were discarded. Subsequently, utilizing the converged slices, the 2D PMF surfaces (Fig 2) and their standard deviations (S17B–S17D Fig) were computed using WHAM. The statistics of the exchange ratios between neighboring US replicas in HREX simulations are shown in S18 Fig. Overall, the exchange ratios are greater than 20% in 2D HREX simulations, indicating enough sampling efficiencies (S18 Fig).

Cell lines

HEK 293T, GHOST, and HeLa cells were maintained at 37°C in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated bovine serum, 1% penicillin-streptomycin, and 1% glutamine in 5% CO₂. Human PBMCs were isolated from leukopheresis packs obtained from the Central Blood Bank (Pittsburgh, Pennsylvania) via Ficoll-Paque Plus (GE Healthcare, Marlborough, Massachusetts) density gradient centrifugation, following manufacturer’s instructions, and stimulated with 50 U/mL recombinant interleukin-2 (IL-2, Thermo Fisher, Waltham, Massachusetts) and 5 μg/mL phytohaemagglutinin (PHA, Thermo Fisher) for 48 to 72 h prior to infection. CD14+ monocytes were purified from PBMCs via CD14 MicroBeads (Miltenyi, Auburn, California) magnetic cell sorting, following manufacturer’s instructions, and differentiated into macrophages with 50 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF, R&D Systems, Minneapolis, Minnesota) for 6 d prior to infection.

Isolation of HIV-1 capsids for AFM measurements

HIV-1 pseudovirions were used to isolate capsids as previously described [55,56]. Briefly, approximately 10⁶ human embryonic kidney (HEK) 293T cells were transfected with 2.5 μg of ΔEnv IN- HIV-1 plasmid (DHIV3-GFP-D116G) [17] using 10 μg of polyethylenimine (PEI, branched, MW approximately 25,000, Sigma-Aldrich, St. Louis, Missouri). After 20 h, the medium was replaced with fresh medium. After 6 h, the supernatant was harvested, centrifuged at 1,000 rpm for 10 min, and filtered through a 0.45-μm-pore size filter. The virus-containing supernatant was concentrated by ultracentrifugation in an SW-28 rotor (25,000 rpm, 2 h, 4°C) using OptiPrep density gradient medium (Sigma-Aldrich, Darmstadt, Germany). Virus containing fractions were collected, mixed with 10 ml of TNE buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA (pH 7.4)) and added to 100-kDa molecular mass cutoff Vivaspin 20 centrifugal concentrators (100,000 MWCO, Sartorius AG, Germany). The mixture was centrifuged twice at 2,500 × g for 25 to 30 min at 4°C, until the supernatant level in the concentrators reached 200 to 300 μL.

Capsids were isolated from concentrated virus-containing material using a previously described protocol [57] with modifications. Briefly, approximately 40 μL of purified HIV-1 pseudovirions was mixed with an equal amount of 1% Triton-X diluted in 100 mM 3-(N-morpholino) propane sulfonic acid MOPS buffer (pH 7.0) and incubated for 2 min at 4°C. The mixture was centrifuged at 13,800 × g for 8 min at 4°C. After removing the supernatant, the pellet was washed twice by adding approximately 80 μl of MOPS buffer and centrifuging at 13,800 × g for 8 min at 4°C. The pellet was resuspended in 10 μl of MOPS buffer.

AFM measurements and analysis

AFM measurements and analysis were performed as previously described[17,28]. Briefly, 10 μL of isolated HIV-1 capsids resuspended in MOPS buffer was incubated for 30 min at room temperature on hexamethyldisilazane (HMDS)-coated microscope glass slides. Measurements were carried out in MOPS buffer, without sample fixation. Every experiment was repeated at least 3 times, each time with independently purified HIV-1 capsids. IP₆ was provided from the James laboratory as a generous gift. Measurements were carried out with a JPK Nanowizard Ultra-Speed atomic force microscope (JPK Instruments, Berlin, Germany) mounted on an inverted optical microscope (Axio Observer; Carl Zeiss, Heidelberg, Germany) using silicon nitride probes (mean cantilever spring constant, kcant = 0.12 N/m, DNP, Bruker, Camarillo, California). Height topographic and mechanical map images were acquired in quantitative imaging (QI) mode, at a rate of 0.5 lines/s and a loading force of 300 pN. All measurements were carried out in MOPS buffer which contained 100 μM IP₆ or mellitic acid (Sigma) when relevant.

Capsid stiffness was obtained by the nanoindentation method as previously described [17,28,57]. Briefly, the stiffness value of each capsid was determined by acquiring approximately 400 force-distance (F-D) curves. To determine the stiffness value of capsids, 20 F-D curves at a rate of 20 Hz at each of 24 different points on the capsid surface were determined. To confirm that the capsid remained stable during the entire indentation experiment, we monitored individual measured point stiffness as a histogram (S19A Fig) and as a function of the measurement count (S19B Fig). Samples whose point stiffness values decreased consistently during experimentation were discarded, since they underwent irreversible deformation.

The maximum indentation of the sample was 4 nm, which corresponds to a maximum loading force of 0.2 to 1.5 nN. Prior to analysis, each curve was shifted to set the deflection in the noncontact section to zero. The set of force distance curves was then averaged (S19C Fig). From the slope of the averaged F-D curve, measured stiffness was derived mathematically. The stiffness of the capsid was computed using Hooke's law on the assumption that the experimental system may be modeled as 2 springs (the capsid and the cantilever) arranged in series. The spring constant of the cantilever was determined during experiment by measuring thermal fluctuation [58]. To reduce the error in the calculated point stiffness, we chose cantilevers such that the measured point stiffness was <70% of the cantilever spring constant. Data analysis was carried out using MATLAB software (The Math Works, Natick, Massachusetts).

Viruses

Mutations were generated in previously described single-round plasmid derivatives of HIV-1_NL4-3 (pNLdE-luc) [59] that encode for luciferase via the QuikChange site-directed PCR mutagenesis kit (Agilent, Santa Clara, California). Resulting plasmid DNAs were verified by Sanger sequencing. Virus was produced in HEK 293T cells via transfection of the proviral plasmids with pL-VSV-G [60] and, for imaging assays, pcDNA5-TO-Vpr-mRuby3-IN [61] using Lipofectamine 2000 (Thermo Fisher). Supernatants were harvested after 48 h, centrifuged to remove cells, and stored in aliquots at −80°C. The infectivity of HIV-1 stocks normalized by p24 content (XpressBio, Frederick, Maryland) was assayed by infection of GHOST cells as previously described [62].

ERT with WT and CA-mutant protein for AFM analysis

Reverse transcription was induced in isolated capsids attached to HMDS-coated microscope glass slides. To initiate reverse transcription, MOPS buffer was replaced with reverse transcription buffer (100 μM dNTPs and 1 mM MgCl₂ in 100 mM MOPS buffer (pH 7.0)) [29]. All measurements were carried out at room temperature (23 to 25°C).

HIV-1 CA retention assay

Equal p24 amounts of virus containing mRuby3-IN packaged in trans were diluted in STE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA (pH 7.4)) supplemented with 100 μM IP₆ and adhered to 35 mm glass-bottom dishes (MatTek, Ashland, Massachusetts) coated with Cell-Tak (Corning) for 30 min at 37°C following manufacturer’s instructions. Samples were washed with PBS and briefly permeabilized with 0.02% Triton X-100 (Thermo Fisher, Waltham, Massachusetts) for 30 s, then again washed with PBS and fixed with 2% paraformaldehyde (PFA). For IP₆ studies, samples that had been adhered to glass and briefly permeabilized as above were additionally incubated for 2 h at 37°C in STE buffer with or without 100 μM IP₆. To address the possibility that the preincubation brief permeabilization was transient and the virus membrane had resealed during the 2-h incubation (thereby trapping loose CA within the virus membrane), samples were briefly permeabilized again after incubation to ensure loose CA was able to disperse, prior to washing and fixation. Fixed samples were permeabilized with 0.1% Triton X-100, blocked with normal donkey serum, immunostained for CA/p24 using mouse anti-CA monoclonal antibody 24–4 (Santa Cruz, Dallas, Texas) and donkey anti-mouse IgG-Cy5, and mounted with coverslips. Confocal imaging was performed on a Nikon A1 laser scanning confocal microscope, with 6 fields imaged per sample. CA staining and mRuby3-IN-containing particles were modeled and enumerated with Imaris imaging analysis software (Bitplane, Zurich, Switzerland). The number of stained CA puncta per field was normalized to the number of mRuby3-IN particles, which were taken as representing intact capsids. The normalized number of CA puncta was reported for each condition (with or without permeabilization) or reported as the percentage of non-permeabilized puncta retained upon permeabilization.

Infectivity assays and measurement of reverse transcripts and 2-LTR circles

Cells were plated overnight in 24-well or 96-well plates and challenged with virus at equal amounts of p24. Virus infectivity was determined by luciferase production (Promega) after 48 h using a Synergy2 Multi-Detection Microplate Reader (BioTek, Winooski, Vermont)

HeLa cells were plated in 6-well plates and infected with virus at equal amounts of p24 after treatment with DNaseI (Roche, Indianapolis, Indiana) for 30 min at 37°C. Control infections were performed in the presence of 25 nM rilpivirine. After 24 h, cells were washed with PBS, trypsinized, and pelleted, and DNA was extracted using the Blood Mini Kit (Qiagen, Germantown, Maryland). Early (RU5) and late (gag) HIV-1 reverse transcripts and 2-LTR circles were measured by qPCR as previously described [16]. Values obtained in the presence of rilpivirine were subtracted from experimental values to determine levels of viral reverse transcription and 2-LTR circle formation.

Statistics of infectivity assays, quantitative PCR, and capsid stability assay

Results were analyzed for statistical significance by 2-sided Student t test (infectivity assays and quantitative PCR) or 1-way ANOVA with Tukey multiple comparisons test (capsid stability assay) with Prism software (GraphPad, San Diego, California). A p-value of less than or equal to 0.05 was used to indicate statistical significance.

TEM of viruses

Virus produced from HEK 293T cells was centrifuged to remove cells, filtered through a 0.45 μm Polyethersulfone (PES) syringe filter (Millipore, Burlington, Massachusetts), transferred into 25 × 89 mm polyallomer ultracentrifuge tubes (Beckman Coulter, Brea, California), and ultracentrifuged in an Optima XL-100K ultracentrifuge (Beckman Coulter) using an SW28 rotor at 25,500 rpm (117,250 × g) for 2.5 h at 4°C. Following aspiration of supernatant, the pellet was fixed in FGP fixative (1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% picric acid in 0.1 M sodium cacodylate buffer (pH 7.4)) for 2 h at room temperature and stored at 4°C. Ultrathin sections (60 nm) were cut on a Reichert Ultracut-S microtome, transferred to copper grids stained with lead citrate, and observed using a JEOL 1200EX microscope with an AMT 2k charge-coupled-device camera. Images captured at 30,000× magnification were visually inspected to classify viral particles as mature, immature, eccentric, or containing 2 apparent capsids, and over 100 particles were counted per virus preparation; representative TEM images are shown in S20 and S21 Figs and summarized in S8 Table. Statistical significance versus matched WT morphology was assessed using 2-way ANOVA with Sidak multiple comparisons test with GraphPad Prism software.

Preparation of recombinant CA assemblies

U-¹³C,¹⁵N labeled CA K25N and K25A mutants were isolated and purified using the protocol reported previously with minor modifications [16]. Pure proteins were assembled as reported previously [17] with minor modifications. First, the proteins were dialyzed overnight against 50 mM MES buffer (pH 6.0) containing 300 mM NaCl. The dialyzed proteins were concentrated to 40 mg/mL and diluted to 1:1 volumetric ratio with assembly buffer (50 mM MES (pH 6.0), 600 μM NaCl). The final assembly was carried at room temperature by 1:1 volumetric dilution with 1,800 μM inositol hexaphosphate (IP₆) (pH 6.0). CA assemblies were pelleted after the incubation for 1 h and stored at 4°C.

Preparation of recombinant CA-SP1-NC assemblies

Protein was purified and assembled as previously described by the authors [57].

Transmission electron microscopy of CA assemblies

After assembly, a small aliquot of each sample was removed and immediately stained with 2% uranyl acetate on copper grids. Images were collected using the TALOS F200C at the Keck Center for Advanced Microscopy and Microanalysis of Interdisciplinary Science and Engineering (ISE) Laboratory of the University of Delaware.

MAS NMR Spectroscopy of CA assemblies

MAS NMR experiments were performed on a Bruker 20.0 T narrow bore AVIII spectrometer. The Larmor frequencies of ¹H, ¹³C, and ¹⁵N were 850.4, 213.8, and 86.2 MHz, respectively.

The U-¹³C, ¹⁵N-CA WT and K25A conical assemblies were packed into 3.2 mm thin wall MAS NMR rotors, and ¹³C-¹³C CORD data sets were acquired using a 3.2 mm E-Free HCN probe. The sample temperature was 5 ± 1°C controlled by the Bruker VT controller. The MAS frequency was 14.000 ± 0.001 kHz controlled by the Bruker MAS-3 controller. The 90° pulse lengths were 2.9 μs (¹H) and 4.3 μs (¹³C). The cross-polarization contact time was 1 ms; a linear 90% to 110% amplitude ramp of was applied on ¹H; the center of the ramp was at 82 kHz and Hartmann-Hahn matched to the first spinning sideband; ω_RF = 54 kHz was applied on the ¹³C channel. SPINAL-64 ¹H decoupling [63] (ω_RF = 86 kHz) was applied during t₁ and t₂ periods. The CORD mixing time was 50 ms. The spectra were acquired as a 2,048 × 864 (t₂ × t₁) points complex matrix to the final acquisition times of 15.98 and 10.29 ms, respectively. States-TPPI protocol [64] was used for frequency discrimination in the indirect dimension. A total of 156 transients were added up for each FID; the recycle delay was 2 s.

The U-¹³C, ¹⁵N-CA K25N conical assemblies were packed into a 1.3 mm MAS NMR rotor, and ¹³C-¹³C RFDR spectrum was acquired using a 1.3 mm HCN probe. The sample temperature was 5 ± 1°C controlled by the Bruker VT controller. The MAS frequency was 60.000 ± 0.001 kHz controlled by the Bruker MAS-3 controller. The 90° pulse lengths were 1.4 μs (¹H) and 2.85 μs (¹³C), with T_CP of 1 ms; a linear 90% to 110% amplitude ramp of was applied on ¹H; the center of the ramp was at 100 kHz and Hartmann-Hahn matched to the first spinning sideband; ω_RF = 20 kHz was applied on the ¹³C channel. TPPM ¹H decoupling [65] (ω_RF = 10 kHz) was applied during t₁ and t₂ periods. The spectrum was acquired as a 2,048 × 800 (t₂ × t₁) points complex matrix to the final acquisition times of 15.98 and 9.84 ms, respectively. States-TPPI was used for frequency discrimination in the indirect dimension. A total of 256 transients were added up for each FID; the recycle delay was 2 s.

The spectra were processed with 50° shifted squared sine-bells in both dimensions and zero-filled to at least twice the number of points in the indirect dimension. The processed spectra were analyzed in NMRFAM-Sparky [66]. Chemical shifts of CA NL_4-3 tubular assemblies [67,68] were used for data analysis (BMRB: 30741; PDBID: 6X63).