Sedimentation velocity experiments were conducted using an Optima Analytical Ultracentrifuge (Beckman Coulter, Brea, CA, USA) at a temperature of 20 °C. Protein was diluted in 20 mM Tris, pH 8.0, 150 mM NaCl, 10% (v/v) glycerol, 1 mM TCEP and where relevant, 100 μM RK-33 or an equivalent % DMSO was added prior to centrifugation. In total, 380 μL of sample and 400 μL of reference solution were loaded into a conventional double sector quartz cell and mounted in a Beckman 8-hole An-50 Ti rotor (Beckman Coulter, Brea, CA, USA). Samples were centrifuged at a rotor speed of 40,000 rpm and the data were collected continuously at multiple wavelengths (290 and 410 nm). Solvent density (1.0331 g/mL at 20 °C) and viscosity (1.385 cP at 20 °C), as well as estimates of the partial specific volume (0.7247 mL/g for DDX3X at 20 °C), were calculated using the program SEDNTERP (Durham, NH, USA) [24 ]. Sedimentation velocity data were fitted to a continuous size (c(s)) distribution model using the program SEDFIT (Bethesda, MD, USA) [25 (link)].
Optima analytical ultracentrifuge
Manufactured by Beckman Coulter
Sourced in United States
The Optima Analytical Ultracentrifuge is a laboratory instrument designed for the analysis of macromolecular structures and interactions. The core function of this equipment is to subject samples to high-speed centrifugation, allowing for the separation and analysis of molecules based on their sedimentation properties.
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10 protocols using optima analytical ultracentrifuge
1
Sedimentation Velocity Analysis of DDX3X
2
Analytical Ultracentrifugation of Protein Complexes
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Analytical ultracentrifugation sedimentation velocity experiments were performed with an Optima Analytical Ultracentrifuge (Beckman-Coulter Inc.) using a An-50 Ti rotor. Samples were spun at 40,000 rpm at 20 °C. Samples were prepared by thoroughly dialyzing against 30 mM HEPES, pH 7.8, 100 mM KCl, 10% glycerol, and 1 mM TCEP. Sample (380 μL) and buffer (400 μL) were filled in each sector of 2-sector charcoal quartz cells. Absorbance was monitored at 280 nm for samples using absorbance optics and scans were obtained at 3 min intervals. The density and viscosity of the buffer at 20 °C was calculated using SEDNTERP. The continuous distribution sedimentation coefficient (c(s)) model was used to fit the AUC data using SEDFIT.63 (link)
3
Analytical Ultracentrifugation Sedimentation Velocity
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Analytical ultracentrifugation sedimentation velocity experiments were performed with an Optima Analytical Ultracentrifuge (Beckman-Coulter Inc.) using a An-50 Ti rotor. Samples were spun at 40,000 rpm at 20 °C. Samples were prepared by thoroughly dialyzing against 30 mM HEPES, pH 7.8, 100 mM KCl, 10% glycerol, and 1 mM TCEP. Sample (380 μL) and buffer (400 μL) were filled in each sector of 2-sector charcoal quartz cells. Absorbance was monitored at 280 nm for samples using absorbance optics and scans were obtained at 3 min intervals. The density and viscosity of the buffer at 20 °C was calculated using SEDNTERP. The continuous distribution sedimentation coefficient (c(s)) model was used to fit the AUC data using SEDFIT66 (link).
4
SV-AUC Analysis of TDP-43 NTD-NLS
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SV-AUC experiments on TDP-43 NTD-NLS:ΔIBB-importin α1 heterodimer was performed at concentrations ranging 1–2 mg/mL in 20 mM Tris-HC pH 7.5, 150 mM NaCl, and 3 mM β-ME. Centrifugation was performed at 40,000 rpm at 6°C using a Beckman XLA Analytical Ultracentrifuge (Beckman-Coulter, Brea, CA). SV-AUC experiments on TDP-43 NTD-NLS and Importin α/β were performed at 20°C with an Optima analytical ultracentrifuge (Beckman-Coulter, Brea, CA) and a TiAn50 rotor with two-channel charcoal-filled Epon centerpieces and sapphire windows, using both absorbance and interference optics. Data were collected with detection at 260, 280, & 295 nm, as well as interference optics. TDP-43 NTD-NLS analyses were performed in 20 mM Tris-HCL pH 7.5, 150 mM NaCl, and 3 mM β-ME, and Importin α/β-NTD-NLS analyses were performed in 20 mM Tris-HCL pH 8.0, 50 mM NaCl, and 2 mM DTT. Complete sedimentation velocity profiles were recorded every 30 s at 40,000 rpm. Data were fit using the c(S) implementations of the Lamm equation as implemented in the program SEDFIT (Schuck, 2000 (link)). The partial specific volume (ῡ), solvent density (ρ), and viscosity (η) were derived from chemical composition by SEDNTERP (Laue et al., 1992 ). All S values calculated were corrected to s20,w values. Figures were created using the program GUSSI (Brautigam, 2015 (link)).
5
Analytical Ultracentrifugation Characterization
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Purified protein samples were thawed on ice and the buffer was replaced using G-25 columns (PD MiniTrap G-25, GE Healthcare) with 25 mM Tris–HCl pH 8, 300 mM NaCl and 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP, Sigma). Appropriate amounts of the different proteins were mixed and diluted in the same buffer with a final volume of 300 μl that was charged into two sector analytical ultracentrifugation cells. The centrifugation was performed for 16 h in an Optima analytical ultracentrifuge (Beckman-Coulter) with an 8-hole AnTi50 rotor, at 42 000 rpm and 20°C, measuring the optical density at 280 nm in order to determine the sedimentation profile of the protein or complex. The data were analyzed with the SedFit software (NIH) using a diffusion deconvoluted continuous sedimentation coefficient distribution c(s) model with one discrete species (60 (link)).
The partial specific volume (Vbar) of the proteins was estimated by the software Sednterp (61 ) based on the amino acid sequence. This software was also used for the calculation of the density and viscosity of the buffer used throughout the experiments. The experiments were reproduced two times and produced identical results.
The partial specific volume (Vbar) of the proteins was estimated by the software Sednterp (61 ) based on the amino acid sequence. This software was also used for the calculation of the density and viscosity of the buffer used throughout the experiments. The experiments were reproduced two times and produced identical results.
6
SV-AUC Analysis of TDP-43 NTD-NLS
7
Quantifying AAV Capsid Composition
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Using a Beckman Coulter Optima Analytical Ultracentrifuge, a sedimentation velocity assessment was performed with interference data of 100 scans collected and analyzed using Sedfit to generate a c(s) distribution plot of empty and full capsid peaks. The c(s) peaks corresponding to the AAV particles between 55 and 105 S20w were integrated to determine relative abundance of empty and full capsids (Supplementary Table S8 ).
The vector was single stranded. The SMN1 transgene was the native SMN1 sequence and not codon optimized.
The vector was single stranded. The SMN1 transgene was the native SMN1 sequence and not codon optimized.
8
Sedimentation Velocity Analysis of Viral Samples
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SV-AUC was performed following the method of Burnham et al.79 (link) Briefly, the AC3 samples were diluted to a final optical density 260 (OD260; 0.2), which corresponds to ∼ 2E+12 gc/mL, in 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, 0.005% PF-68 (pH 8.1), and a 0.4-mL sample was loaded into a Beckman Charcoal-Epon two-sector cell with 12-mm centerpieces and either sapphire or quartz windows. SV experiments were performed using an Optima analytical ultracentrifuge equipped with a scanning ultraviolet-visible optical system (Beckman Coulter, Indianapolis, IN, USA) All experiments were performed at 20°C after at least 1 h of equilibration after the rotor reached 20°C. A rotor speed of 20,000 rpm, UV detection at 260 nm, and a scan frequency of 60 s were used for a total of 150 scans. The data were analyzed using Sedfit (Peter Schuck, NIH) fitting the data to a continuous c(s) distribution from 0S–200S. A resolution of 300 points per distribution and a confidence level of 0.95 were applied to all fits. Baseline, radial independent noise, and time-independent noise were fit, while the meniscus and bottom positions were set manually. The c(s) distributions were imported into Origin 2018 (OriginLab, Northampton, MA, USA) for peak integrations and graph generation.
9
Sedimentation Analysis of Peptides
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Sedimentation velocity and sedimentation equilibrium experiments were performed using a Beckman Coulter Optima Analytical Ultracentrifuge. An-60 Ti rotor and standard 12 mm epon centerpieces were used for both experiments. All measurements were done at a temperature of 293 K and in 0.2 M NaCl in order to screen electrostatic repulsions between molecules.
Sedimentation velocity data were collected at 50 000 rpm. PGA and PLL were measured in absorbance mode at a wavelength of 230 nm and at a peptide concentration of 220 mg L À1 . Data analysis was carried out by both Sedfit 30 and Ultrascan 31 analysis software.
Sedimentation equilibrium experiments were performed at three speeds: 20 000, 25 000, and 30 000 rpm. The absorbance mode, wavelength of 230 nm, and concentration of 220 mg L À1 were used for both PGA and PLL samples. The obtained data were analyzed by Sedphat 30 software. Detailed information about experimental design and data analysis procedures can be found in our previous article. 32
Sedimentation velocity data were collected at 50 000 rpm. PGA and PLL were measured in absorbance mode at a wavelength of 230 nm and at a peptide concentration of 220 mg L À1 . Data analysis was carried out by both Sedfit 30 and Ultrascan 31 analysis software.
Sedimentation equilibrium experiments were performed at three speeds: 20 000, 25 000, and 30 000 rpm. The absorbance mode, wavelength of 230 nm, and concentration of 220 mg L À1 were used for both PGA and PLL samples. The obtained data were analyzed by Sedphat 30 software. Detailed information about experimental design and data analysis procedures can be found in our previous article. 32
10
Characterization of CBM-AQ12-CBM Protein
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Cloning, expression and purification of the CBM-AQ12-CBM was carried out according to our earlier study. 14 (link) Determination of the anisotropy (deviation from spherical shape) of the protein was conducted by a Beckman Coulter Optima Analytical Ultracentrifuge.
Sedimentation velocity experiment were performed at 50000 rpm and at 20 o C. As a buffer we used 0.1 M of NaCl, concentration of the CBM-AQ12-CBM, commonly referred to also as CBM-eADF3-CBM, 14 protein was 0.5 mg/ml. Sample was measured by UV/Vis absorbance optics at 280 nm. Data analysis were performed by Ultrascan III version 4.0 revision 2528 (http://www.ultrascan.aucsolutions.com). Partial specific volume was determined from the sequence and is 0.6994. Noise reduction was conducted by two dimensional spectrum analysis. 81 (link) We used Genetic Algorithm for further refinement and regularization. 82 (link) More detailed information about the experiment setups and data treatment procedures is available in Ref. 83 (link) . In presented data values, the error estimate corresponds to a 95% confidence interval.
The frictional ratio of protein constructs were estimated using the UltraScan Solution Modeler (US-SOMO). 84 (link)
Sedimentation velocity experiment were performed at 50000 rpm and at 20 o C. As a buffer we used 0.1 M of NaCl, concentration of the CBM-AQ12-CBM, commonly referred to also as CBM-eADF3-CBM, 14 protein was 0.5 mg/ml. Sample was measured by UV/Vis absorbance optics at 280 nm. Data analysis were performed by Ultrascan III version 4.0 revision 2528 (http://www.ultrascan.aucsolutions.com). Partial specific volume was determined from the sequence and is 0.6994. Noise reduction was conducted by two dimensional spectrum analysis. 81 (link) We used Genetic Algorithm for further refinement and regularization. 82 (link) More detailed information about the experiment setups and data treatment procedures is available in Ref. 83 (link) . In presented data values, the error estimate corresponds to a 95% confidence interval.
The frictional ratio of protein constructs were estimated using the UltraScan Solution Modeler (US-SOMO). 84 (link)
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