The purpose of this post is to demonstrate a first approach to the analysis of multiwavelength kinetic data, like those obtained using stopped-flow data. To practice, we will use data that were acquired during the stopped flow practicals of the
MetBio summer school from the
FrenchBIC. During the practicals, the student monitored the reaction of myoglobin (in its Fe(III) state) with azide, which yields a fast and strong change in the absorbance spectrum of the protein, which was monitored using a diode array. The data is publicly available on
zenodo.
Aims of this tutorial
The purpose of this tutorial is to teach you to use the free software
QSoas to run a simple, multiwavelength exponential fit on the data, and to look at the results. This is not a kinetics lecture, so that it will not go in depth about the use of the exponential fit and its meaning.
Getting started: loading the file
First, make sure you have a working version of QSoas, you can download them (for free)
there. Then download the data files from
zenodo. We will work only on the data file
Azide-1.25mm_001.dat, but of course, the purpose of this tutorial is to enable you to work on all of them. The data files contain the time evolution of the absorbance for all wavelengths, in a
matrix
format, in which each row correpond to a time point and each column to a wavelength.
Start
QSoas, and launch the command:
QSoas> load /comments='"'
Then, choose the
Azide-1.25mm_001.dat data file. This should bring up a horizontal red line at the bottom of the data display, with X values between about 0 and 2.5. If you zoom on the red line with the mouse wheel, you'll realize it is data. The
/comments='"' part is very important since it allows the extraction of the wavelength from the data. We will look at what it means another day. At this stage, you can look at the loaded data using the command:
QSoas> edit
You should have a window looking like this:
The rows each correspond to a data point displayed on the window below. The first column correspond to the X values, the second the Y values, and all the other ones to extra Y columns (they are not displayed by default). What is especially interesting is the first row, which contains a
nan as the X value and what is obviously the wavelength for all the Y values. To tell that QSoas should take this line as the wavelength (which will be the
perpendicular coordinate
, the coordinate of the other direction of the matrix), first close the edit window and run:
QSoas> set-perp /from-row=0
Splitting and fitting
Now, we have a single dataset containing a lot of Y columns. We want to fit all of them simultaneously with a (mono) exponential fit. For that, we first need to split the big matrix into a series of X,Y datasets (because fitting only works on the first Y). This is possible by running:
QSoas> expand /style=red-to-blue /flags=kinetics
Your screen should now look like this:
You're looking at the kinetics at all wavelengths at the same time (this may take some time to display on your computer, it is after all a rather large number of data points). The
/style=red-to-blue is not strictly necessary, but it gives the red to blue color gradient which makes things easier to look at (and cooler !). The
/flags=kinetics is there to attach a label (a
flag
) to the newly created datasets so we can easily manipulate all of them at the same time. Then it's time to fit, with the following command:
QSoas> mfit-exponential-decay flagged:kinetics
This should bring up a new window. After resizing it, you should have something that looks like this:
The bottom of the fit window is taken by the parameters, each with two checkboxes on the right to set them fixed (i.e. not determined by the fitting mechanism) and/or global (i.e. with a single value for all the datasets, here all the wavelengths). The top shows the current dataset along with the corresponding fit (in green), and, below, the residuals. You can change the dataset by clicking on the horizontal arrows or using Ctrl+PgUp or Ctrl+PgDown (keep holding it to scan fast). See the
Z = 728.15 showing that QSoas has recognized that the currently displayed dataset corresponds to the wavelength 728.15. The equation fitted to the data is: $$y(x) = A_\infty + A_1 \times \exp -(x - x_0)/\tau_1$$
In this case, while the \(A_1\) and \(A_\infty\) parameters clearly depend on the wavelength, the time constant of evolution should be independent of wavelength (the process happens at a certain rate regardless of the wavelength we're analyzing), so that the \(\tau_1\) parameter should be common for all the datasets/wavelengths. Just click on the
global
checkbox at the right of the
tau_1 parameter, make sure it is checked, and hit the
Fit
button...
The fit should not take long (less than a minute), and then you end up with the results of the fits: all the parameters. The best way to look at the non global parameters like \(A_1\) and \(A_\infty\) is to use the
Show Parameters
item from the
Parameters
menu. Using it and clicking on
A_inf too should give you a display like this one:
The
A_inf parameter corresponds to the spectum at infinite time (of azide-bound heme), while the
A_1 parameter corresponds to the difference spectrum between the initial (azide-free) and final (azide-bound) states.
Now, the fit is finished, you can save the parameters if you want to reload them in a later fit by using the
Parameters
/
Save
menu item or export them in a form more suitable for plotting using
Parameters
/
Export
(although QSoas can also display and the parameters saved using
Save
). This concludes this first approach to fitting the data. What you can do is
How to read the code above
All the lines starting by
QSoas> in the code areas above are meant to be typed into the QSoas command line (at the bottom of the window), and started by pressing
enter at the end. You must remove the
QSoas> bit. The other lines (when applicable) show you the response of QSoas, in the terminal just above the command-line. You may want to play with the
QSoas tutorial to learn more about how to interact with QSoas.
About QSoas
QSoas is a powerful open source data analysis program that focuses on flexibility and powerful fitting capacities. It is released under the
GNU General Public License. It is described in
Fourmond, Anal. Chem., 2016, 88 (10), pp 5050 5052. Current version is 3.1. You can freely (and at no cost) download its source code or precompiled versions for MacOS and Windows
there. Alternatively, you can clone from the
GitHub repository.
Contact: find my email address
there, or contact me on
LinkedIn.
Holger Levsen: Welcome, David, thanks for taking the time to talk with us today. First, could you briefly tell me about yourself?
David: Sure! I m David A. Wheeler and
I work for the 
My impression of that city from couple of visits at that point in time where they were still more tongas (horse-ridden carriages), an occasional two wheelers and not many three wheelers. Although, it was one of the more turbulent times as lot of agitation for worker rights were happening around that time and a lot of 
So the last three stories I found the most intriguing.
The first one is titled Man on the Beach. Apparently, a gentleman goes to one of the beaches, a sort of lonely beach, hails a taxi and while returning suddenly dies. The Taxi driver showing good presence of mind takes it to hospital where the gentleman is declared dead on arrival. Unlike in India, he doesn t run away but goes to the cafeteria and waits there for the cops to arrive and take his statement. Now the man is in his early 40s and looks to be fit. Upon searching his pockets he is found to relatively well-off and later it turns out he owns a couple of shops. So then here are the questions ?
What was the man doing on a beach, in summer that beach is somewhat popular but other times not so much, so what was he doing there?
How did he die, was it a simple heart attack or something more? If he had been drugged or something then when and how?
These and more questions can be answered by reading the story Man on the Beach .
2. The death of a photographer Apparently, Kurt lives in a small town where almost all the residents have been served one way or the other by the town photographer. The man was polite and had worked for something like 40 odd years before he is killed/murdered. Apparently, he is murdered late at night. So here come the questions
a. The shop doesn t even stock any cameras and his cash box has cash. Further investigation reveals it is approximate to his average takeout for the day. So if it s not for cash, then what is the motive ?
b. The body was discovered by his cleaning staff who has worked for almost 20 years, 3 days a week. She has her own set of keys to come and clean the office? Did she give the keys to someone, if yes why?
c. Even after investigation, there is no scandal about the man, no other woman or any vices like gambling etc. that could rack up loans. Also, nobody seems to know him and yet take him for granted till he dies. The whole thing appears to be quite strange. Again, the answers lie in the book.
3. The Pyramid Kurt is sleeping one night when the telephone rings. The scene starts with a Piper Cherokee, a single piston aircraft flying low and dropping something somewhere or getting somebody from/on the coast of Sweden. It turns and after a while crashes. Kurt is called to investigate it. Turns out, the plane was supposed to be destroyed. On crash, both the pilot and the passenger are into pieces so only dental records can prove who they are. Same day or a day or two later, two seemingly ordinary somewhat elderly women, spinsters, by all accounts, live above the shop where they sell buttons and all kinds of sewing needs of the town. They seem middle-class. Later the charred bodies of the two sisters are found :(. So here come the questions
a.Did the plane drop something or pick something somebody up ? The Cherokee is a small plane so any plane field or something it could have landed up or if a place was somehow marked then could be dropped or picked up without actually landing.
b. The firefighter suspects arson started at multiple places with the use of petrol? The question is why would somebody wanna do that? The sisters don t seem to be wealthy and practically everybody has bought stuff from them. They weren t popular but weren t also unpopular.
c. Are the two crimes connected or unconnected? If connected, then how?
d. Most important question, why the title Pyramid is given to the story. Why does the author share the name Pyramid. Does he mean the same or the original thing? He could have named it triangle. Again, answers to all the above can be found in the book.
One thing I also became very aware of during reading the book that it is difficult to understand people s behavior and what they do. And this is without even any criminality involved in. Let s say for e.g. I die in some mysterious circumstances, the possibility of the police finding my actions in last days would be limited and this is when I have hearing loss. And this probably is more to do with how our minds are wired. And most people I know are much more privacy conscious/aware than I am.
This post describes how I ve put together a simple static content server for
kubernetes clusters using a Pod with a persistent volume and multiple
containers: an sftp server to manage contents, a web server to publish them
with optional access control and another one to run scripts which need access
to the volume filesystem.
The sftp server runs using