Figure 1.
Focal scans in z-direction perpendicular to the plane of rotation into EGFP samples at various concentrations, taken radially in the middle of the solution column.
The focal scans were normalized (division by the maximum signal in of each focal scan) to better compare their shape at different concentrations.
Figure 2.
Signal intensity as a function of radius and focal depth in the vicinity of the meniscus (A) and bottom (B).
Shown are the signals of EGFP at 100-fit meniscus position from analysis of the high-speed SV experiment is shown after correction for rotor stretch (red lines in the axes planes; measured signals at the meniscus position are shown as blue circles connected with a red line).
Figure 3.
Radial gradients of signal magnification produce sloping solution plateaus.
Shown are FDS-SV data of 135 nM EGFP acquired at a focal depth of 989 μm. (A) Experimental scans (symbols) and best-fit model (solid lines) using a model incorporating TI noise and a radial magnification gradient with best-fit value of dε/dr = 0.28, leading to a best-fit rmsd of 1.79 counts. The TI noise is shown as black line. The sedimentation model is c(s), resulting in a single peak at 2.59 S with frictional ratio 1.39, corresponding to an apparent molecular weight of 31.9 kDa. Residuals are shown in the lower panels, as residuals bitmap and overlay plot. (B) Analogous representation of the same data modeled without radial magnification gradient, which leads to a curved TI profile partially compensating for the sloping plateau. The rmsd of the best fit is 3.42 counts. The peak s-value of c(s) in this fit is 2.65 S, with a frictional ratio of 1.67 corresponding to an apparent molecular weight of 43.3 kDa. (C) Sedimentation coefficient distribution c(s) corresponding to the correct model show in Panel A.
Figure 4.
Dependence of the radial magnification gradient on the focal depth.
Shown are the best-fit values of dε/dr for a sample of 27.4 nM EGFP.
Figure 5.
Illustration of the temporal magnification changes.
FDS-SV data of EGFP at a concentration of 664 nM were acquired at a focal point 3955 μm. (A) The data (crosses) were fitted with a single species model (lines) incorporating a radial gradient of signal magnification dε/dr with a best-fit value of 0.0079 cm−1, and a temporal drift dε/dt of 0.0127 h−1. (B) Based on the best-fit model of Panel A, boundary profiles were calculated for the same data with identical model parameters but eliminating the temporal drift, setting dε/dt = 0. No further fit was done in Panel B, except an adjustment of the macromolecular concentration parameter. In order to highlight the difference in the boundary shapes, the black bar reflects the measured radial dilution (A), whereas the red bar reflects the radial dilution in the absence of signal drifts based purely on geometry of sedimentation (B). The residuals reflect the difference between the data and the model without temporal drift correction.
Figure 6.
Comparison of the effect of beam truncation close to the bottom of the cell at different focal depths.
Shown are the experimental FDS-SV data of 1921 nM EGFP (symbols, only every 2nd scan shown), along with best-fit models based on Eq. 3 and convolution Eq. 4 (lines). Focal depths are 989 μm (A), 5055 μm (B), and 8000 μm (C). The graphics in Panel D illustrates how smaller beam cross-sections (grey), centered at the same radius, are obscured to a different extent by the bottom of the cell (black), thus leading to a partial loss of intensity.
Figure 7.
The dependence of the best-fit beam radius δ (Eq. 4) on focal depth (circles).
The solid line is a linear fit, leading to an apparent beam angle of 5.8°.
Figure 8.
Fit of FDS-SV data at 3258 nM EGFP acquired at a focal depth of 8000 μm.
For clarity only 3rd scan and 3rd data point are shown. (A) Fit and residuals of a model without any FDS-specific corrections with standard c(s), allowing for TI noise (black line). The rmsd of the fit is 17.61 counts. (B) Fit with the same c(s) sedimentation model but additionally including the corrections for radial magnification gradients with best-fit dε/dr = 0.0071 cm−1, temporal intensity drifts with best-fit dε/dt of 2.44%/h, a shadow at the bottom of the cell with radius δ = 0.183 cm, and a radial convolution of σ = 0.03 cm. The rmsd of this fit is 4.96 counts. (C) Sedimentation coefficient distributions resulting from the fit in (A) shown in blue as dotted line, and from the fit in (B) shown in purple as solid line.
Figure 9.
Measured signal increment ε*(c) as a function of EGFP concentration and focal depth at a photomultiplier voltage setting of 32%.
The observed boundary amplitudes after integration of c(s) were normalized with regard to a gain setting of 1 and with regard to loading concentration to calculate the specific signal per nM concentration at different conditions. A surface and contour plot (A) and overlay (B) are shown for the same data. For clarity, experimental series with different focal depth are highlighted with markers of different color: 989 µm in green (two sets), 2055 µm in light blue, 3055 µm and 3095 µm in red, and 5055 µm in purple, 6055 µm in magenta, 7055 µm in dark blue, and 8000 µm in black (two sets).
Figure 10.
Signal weighted-average sedimentation coefficient sw*(c) as a function of EGFP concentration and focal depth.
sw*-values are based on integration of c(s) and each represent an average of values at different gain setting. A surface and contour plot (A) and overlay (B) are shown for the same data. For clarity, experimental series with different focal depth are highlighted with markers of different color: 989 µm in green (two sets), 2055 µm in light blue, 3055 µm and 3095 µm in red, and 5055 µm in purple, 6055 µm in magenta, 7055 µm in dark blue, and 8000 µm in black (two sets).
Figure 11.
Apparent molecular weights as a function of EGFP concentration and focal depth.
Mw*-values are based on integration of c(s) and each represent an average of values at different gain setting. A surface and contour plot (A) and overlay (B) are shown for the same data. For clarity, experimental series with different focal depth are highlighted with markers of different color: 989 µm in green (two sets), 2055 µm in light blue, 3055 µm and 3095 µm in red, and 5055 µm in purple, 6055 µm in magenta, 7055 µm in dark blue, and 8000 µm in black (two sets).