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Equilibrium
sedimentation data analysis software.
Dmitry B. Veprintsev *, Nicholas W. Foster* and Alan R. Fersht, FRS *to
whom correspondence should be addressed
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Double click on the
Ultraspin software, this will open the Data manager page shown below.
Click on File and select open. This will open a new window where you can
select the tutorial data. You can either select a single file and click
ok or select multiple files by clicking multiple files with the Ctrl held
down. Select all the files (ra2 to ra7) and click Ok.
The collected data
will now appear in the main data manager window, a legend appearing on
the right hand side. By clicking on the Serie drop down menu a list of
opened data will also appear.
Select a file to
fit data to, try ra2 by highligthing it in the serie drop down menu. This
is data collected for a monomeric protein so we will try fitting using
the model Spec1_MW_varshift. This will calculate the molecular mass
of our sample whilst floating the baseline value. A fixedshift model could
be used if the baseline has been determined.
As we are doing
a single dataset fit, all the global and local parameters can be set from
the Data fitting window so select Spec1_MW_varshift from the model drop
down menu and click on fit. The following window will now appear.
Click on GetFullLength
to select all data points collected from the file. The range will now appear
in the Region to fit box. If you wish to edit the data to be fitted the
region to be fit may be easily changed from this window. If, as in
this case data has been collected to near to the meniscus, the data to
be edited can be determined as follows. Return to the data manager window
and by carefully clicking on the desired curve to the right of the meniscus
with the left mouse button the data information for that point is displayed
in a new dialogue box. By returning to the fit window you can now simply
enter this number. Any region of the curve may of course be selected.
We now need to enter
the global and local variables for this dataset.
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Global Variables.
M1 - Mass of the protein (9017).
Local Variables Abs0 - Baseline absorbance (0)
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Click Go and data will be fitted. A series of dialgue boxes will be displayed giving indicators of errors and fitting parameters. These will be explained further later. Click ok to these boxes. The apparent molecular mass of the protein is now displayed in the fitting window along with the fit quality results. A fitting curve is overlaid in the Datamanager window and a residual curve is displayed in the bax above.
Lets now try to do
a multi data fit, using all 6 of the data files you have downloaded. In
the main data manager window first select the model Spec1_MW_varshift_Multi
and then click on selectmulltiple and the Multiple dataset window will
open. The left hand box shows files opened, the right hand box shows the
files selected. These can easily be changed by selecting a file and using
the arrow key to move the data. Up to 20 files at one time may be selected.
Select all 6 datasets for fitting. Local variables are now entered into
this window.
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Local Variables.
From and To - Select datapoints for each
dataset.
For ease, use the datapoints indicated in this window, please note the order of the files is not sequential. Global Variables. These variables remain the same as in the single dataset fitting. |
Now click Go in the Data fitting window. Ultraspin will again produce dialogue boxes displaying errors and fitting parameters. Click ok and results for a global fit are displayed in the Data fitting window and the Main data manager window.
Data fitting is easily saved by selected save fit from the File menu on the Data manager window. The Data fitting window can be printed by clicking on print. The Main data manager window may be printed by clicking on File then selecting print. Please note that the print option simply provide a screen dump of the window.
© MRC (UK) Centre for Protein Engineering 2000