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CD Apps© v4.0

Introduction


This piece of software is designed to aid the user in performing a Circular Dichroism (CD) experiment. It aids the initial experimental design and also the collection, processing and graphical presentation of the data. The program requires MS Excel 2007 to be installed on the machine as all the data is stored in an Excel Workbook.
To work in the CD environment it is important to know the absorption properties of the molecules you are working with. As stated by Beer-Lambert's law:
Eqn. 1) A=ε.c.l
Where, A = absorption (no units), ε = absorption coefficient (M-1cm-1), c = concentration (M) and l = the cell path length (cm).
The absorption must be large enough for you to be able to measure a corresponding CD signal (of realistically 1 mdeg or greater). The stronger the signal the better signal to noise and so better data obtained. Optimal CD data is collected at an Absorbance of ~ 0.8. As the absorbance increases above this level, less light is seen by the PMT detector (log scale A = 1, 10% of light seen, A = 2, 1% of light seen etc). To compensate for this the signal obtained is boosted by increasing the HT Voltage of the instrument. This can only be taken so far (usually up to around 650V HT), above this point the CD signal becomes unreliable and is likely to be an "artefact" as the detector becomes increasingly "blind".
Before starting CD Apps you should have an idea of the optimal absorption (~ A = 0.8) of the sample. You should also check the buffer's absorption at the required wavelength range.
When planning a titration measurement using CD Apps, the experiment will have two components (e.g. protein, DNA, RNA, drug etc). The main question to address is "Do these two components bind to each other?" For the purpose of this software one component is called solution A (host), this component is the "constant", present at the start of the titration. The other component is termed solution B (ligand), this component will be titrated into solution A.
For titration measurements, optimal absorption should be ascertained for both solution A and B in the relevant wavelength range. Depending on the results of these, the ratio of both Solution A and B required and the type of titration experiment (fixed or variable – more later) you can work out a concentration for solution A (the constant), to give an absorption A ~ 0.6, then add the absorbance of the highest ratio of solution B, for example A ~ 0.5. These two components should add up to no more than A ~ 1.5.

SETTING UP INITIAL EXPERIMENT PARAMETERS


Load CD Apps by clicking on the "CD Apps" icon. This is normally located in 'W:\cd_programs\CD Apps. You will see the start form shown below. Look through the Terms of use for the CD Apps software. You must agree to these "Terms of Use" when clicking the button.


You will now see the Menu form which can take a few seconds to load as it gathers your visit information based on your Logon (FEDID). Select "Titration Experiments" for titration experiments, "CD Measurement" for non titration experiments and "Utilities" for utilities. "User Documentation" for CDApps manual and the remote access procedure.

CD Titration Experiments

 

  1. When clicking the 'CD Titration' button, the Titration form shown below initially loads looking very blank. When you click on one of the 'Volumes' radio buttons or load experiment parameters the form layout is configured depending on the type of experiment.

  1. If you click on the 'Increasing' or 'Fixed' radio buttons, then most of the form is enabled as shown on the next page. Depending on which radio button you click, certain fields are hidden because they are not needed and also different columns in the datagrid on the right of the form are displayed.

  1. Start by filling out the fields on the left hand side of the form which have a white background (Fields with a grey background are calculated and you won't be able to fill them in).

 

  1. If you need help with the Absorption and Binding Affinity, click on the 'Help' button at the bottom left of the Titration form to display the Absorption form shown on the next page.

  1. If you need help with the extinction coefficient, click on the 'Estimate' button to show the form below.

  1. Once you have filled in the 'Experiment Settings' tab, move onto the 'Spectrometer Settings' tab shown on the right.
  2. The last section to fill in is the Titration data grid. Fill in the values for the 1st 2 columns with up to 28 titration points. Once you have filled in the values for the 1st two columns, click on the 'Calculate Titration Volumes' button for the other columns (and the greyed out fields on the 'Experiment Settings' tab) to be filled in. Each row of the data grid is coloured, with each colour corresponding to one of the B stocks. It is a good idea to save all you settings at this stage by clicking either of the save buttons. An example of a completed Titration form is shown on the next page:







  1. Now that you have finished setting up your experiment parameters you can click on the "'Save' Spreadsheet" button. If this is a new set of Experiment details that you've just typed in, a 'Save As' dialog will appear with the Experiment name as a suggested filename. If you've saved the experiment details at least once before, then the Experiment details will be saved with the previously used name.

Details of the other buttons:
The "'Save As' Spreadsheet" button will bring up a 'Save As' dialog.
Use the 'Load Spreadsheet' button to load previously saved Experiment details.
Use the 'Show/Hide Excel' button to show or hide the instance of Microsoft Excel that may exist if you have previously loaded or saved any Experiment details.
At this stage you will not need the 'Experiment Data' tab or the 'Analyse Experiment Data' buttons.

RUNNING YOUR EXPERIMENT

 

  1. Load up your Experiment parameter workbook created in the previous steps and click on the 'Titration' or 'Non-Titration' sheet depending on your experiment type. This shows all the information required for your experiment. An example titration sheet is shown below:




You can now run your measurements and save the datafile under the filenames generated.

DATA PROCESSING

CD Measurement Experiments

When clicking the 'CD Measurement' button, the CD Measurement form shown below initially loads looking very blank. Select the type of experiment measurement you would like to analyse. When you select one of the Experiment types or load experiment parameters the form layout is configured depending on the type of experiment.

1 to n measurements

  1. Load the spreadsheet that you have saved. Alternatively you could create a new one and fill all the experiment details before loading your datafiles. To do this start by filling out the top fields on the form and once that is done, then start filling in details in the 'Experiment Details' tab. You can add up to 30 compounds and their details, but make sure the 1st one is always the buffer. You can add or remove compounds from the data grid using the 'Add Compound' and 'Remove Compound' button and then reorder them if necessary using the 'Move Up' and 'Move Down' buttons.


  1. Click on the Experiment Data and load your files that you would like to analyse and move them across and save the spreadsheet. Once you have saved it, you can then click "Analyse Experiment Data".


  1. Spectra will be displayed and you can offset the data, restrict the data to remove data due to cut-off of wavelength. Then click "Graph Data".


  1. Three further graphs will be created, i) Offset compound, ii) ??, iii) ??? You can then click "Save Analysis".

UV Denaturation
Please refer to the protocol above in 1 to n measurement.
When you click the "Analyse Experiment Data", there is an additional feature available for principle component analysis "PCA" which you can select. You can also graph to monitor change at a specific wavelength.
There will be 8 further graphs created, i) offset compounds, ii) PCA Scores, iii) PCA Loading, iv) PCA Scree, iv) wavelength monitored, v) ?@wavelength monitored and vi) Folding change @ wavelength change (? A), vii) Folding change @ wavelength change (??).


  1. Thermal Melt
    Single cell measurement
  2. For 'Thermal Melt' type experiments, you also get a 'T Script' tab which allows you to create and save temperature scripts for use to run the sample data acquisition with Olis.


  1. You don't need the 'Experiment Data' tab at this stage. Once you have entered all your experiment parameters, click on either of the save buttons to save these details into an Excel spreadsheet. Below is a screenshot of a filled in form:


To process the thermal denaturation experiment, please refer to the protocol above in 1 to n measurement.
When you click the "Analyse Experiment Data", there is an additional feature available for principle component analysis "PCA" which you can select. You can also graph to monitor change at a specific wavelength.
There will be 8 further graphs created, i) offset compounds, ii) PCA Scores, iii) PCA Loading, iv) PCA Scree, iv) wavelength monitored, v) ?@wavelength monitored and vi) Folding change @ wavelength change (? A), vii) Folding change @ wavelength change (??) as shown in the screen shot below.
To overlay a theoretical model of temperature melting curve on the desired wavelength to be monitored for eg in this case at 209 nm, you can obtain autofitting directly from the software by clicking on the "Auto Fit Boltzmann Curve". If it is not quite right then adjust the parameters of the slope (DX), A1 and A2 or the Tm and click the "Manual Fit Boltzmann Curve" button. To clear all your binding estimates click on the "Clear Binding Estimates" button. Shown below are some binding model estimates:

6-cell turret measurement
6 samples auto-measurement data for temperature studies using the 6-cell turret sample chamber can be analysed by splitting the data collected via the script by right clicking on the data file downloaded as shown below.


On clicking yes button, the datafile will be split into 6 sub-files identified as Tur1_filename; Tur2_filename; ....etc; and Tur6_filename as shown below.

The corresponding buffer and sample subfile ca be carried to the match data files section across as shown below. You must be saved spreadsheet for each unique sample eg. Tur 1_ sampleX before clicking the analyse experiment data button.


Sample measurement can be further analysed as single cell measurement above. Once finished saved the file. You can go back and move across the next subfile for the second sample analysis and save with new filename for the next sample. The process can be repeated until you have processed all 6 sample measurements.

CD Titration Measurements

 

  1. # Once you have finished running your experiment, run CD Apps again and click on the 'Load Spreadsheet' button to load up your experiment parameters. Once they have loaded, click on the 'Experiment Data' tab (Shown on the right).
  2. If you used the Olis instrument then click on the 'Import Experiment Data Files (.OLS)' button or if you have converted the data files to a different format then click on the 'Import Experiment Data Files (.TXT,*.CSV)' button.
  3. In the 'Open Experiment Data Files' dialog select all the data files relevant for this titration experiment and then click on the 'Open' button.
  4. Imported files will be placed in the imported Data Files column on the right.
  5. Move the datafiles across to the Matched Data Files and hit save a spreadsheet. The experimental components will then be displayed giving information of your samples.

  1. Once the files have been imported, each will have an entry in the list box on the left of the form. If you click on any of the entries in the list box then its contents will be displayed in the data grid on the right (as shown below). Also each one will have its own sheet added to the B23 Electronic Workbook. At this stage you can either save the imported data that is now embedded in the B23 Electronic Workbook (using the "'Save' Spreadsheet" or "'Save As' Spreadsheet" buttons) and analyse your data later or move onto the next step.


  1. Move your buffer scan to the top – Save spreadsheet if you have moved your files around. This will enable the "Analyse Experiment Data" button which you need to click next. This will show the "Binding data" form shown below:


When the Binding Data form loads, you are shown an initial graph of the raw data – This data is not normalised. The data used for this graph is displayed in the data grid below it – This applies to all the graphs. There are 3 main steps to this form:

    1. Offset data & restrict data to data where wavelength cut-off appears
    2. Monitor a wavelength and finally
    3. Fit a binding model.

Please note that each part of the form needs to be done in turn starting at the top which is why the "Graph Wavelength to monitor" and "Add binding model" buttons are not displayed as the "Graph data" button hasn't been clicked yet in the screenshot shown above. At any stage you can change values and click any button again, but any processing / output done by a later part of the form will be lost.

  1. To offset the data use the graph shown above and then pick the wavelengths using the 2 combo boxes on the top left. At this stage you can also choose to crop/restrict the data at certain wavelengths by checking the "Restrict data to wavelength range (nm)" check box and picking 2 wavelengths. Once you have set the wavelengths to offset the data at and optionally selected crop wavelengths then click the "Graph Data" button. The data is analysed and 4 more graphs are created with tabs above each graph. They are also added in the B23 Electronic Workbook. The graphs are listed below and 1 shown below:
  2. BHL & Titrations (Raw Data)
  3. A, B & Titrations
  4. Titrations -A -B
  5. Titrations –B

  1. Look at the graphs, decide what wavelength to monitor, then using the either "A, B & Titrations" or "Titrations -B" graph and then just click a point on the graph at the wavelength you want to monitor. This will fill the "Wavelength to monitor (nm)" drop down list box with the selected wavelength and also highlight the selected data point in the data grid below the graph. Once you are happy with the selected wavelength click the "Graph Wavelength to monitor" button. This creates 5 more graphs (228.5nm was selected in the screenshot shown below):
  2. 228.5nm
  3. Δ @ 228.5nm
  4. ΔA @228.5nm
  5. Δε @228.5nm
  6. Binding Stoichiometry

  1. To overlay a theoretical model of binding on the "ΔA @ 228.5nm" graph, please estimate the Kd and Δε (Δε is normally filled in for you), you can either obtain autofitting directly from the software by clicking on the "Auto Fit Binding Curve" It will also gives you a confidence band of deviation from experimental data. Look at the "ΔA @ 228.5nm" graph to see a model of the binding added to the graph. If it is not quite right then adjust the Kd and Δε and click the "Add Binding Model" button again. To clear all your binding estimates click on the "Clear Binding Estimates" button. Shown on the next page are some binding model estimates:

  1. Once you have finished your analysis, click on the "Save Analysis" button to save all the data and graphs in Excel.

Protein Secondary Structure Analysis


Secondary structure analysis can be carried out using the three algorithms available, CONTINLL, CDSSTR and SELCON3
To process the secondary structure analysis, please refer to the protocol above in 1 to n measurement.
When you click the "Analyse Experiment Data", there is other additional feature available such as principle component analysis "PCA", graph to monitor change at a specific wavelength for thermal studies (please refer to the appropriate section above).
Under the SSE as shown below, you can select the algorithm required for the analysis.

You can then select the reference data set appropriate for the wavelength range the samples are collected as in the figure shown below. As a guide, largest dataset are a good start to select (basis set 7, 10). If your sample belongs to the soluble protein, you can select basis set with soluble proteins (basis set 1, 2, 3, 4, 5, 8). If your sample contains a denaturants such as urea or guanidine HCL, you can select a basis set containing denaturated reference samples (basis sets 6 or 9).
CONTINLL and CDSSTR standard deviation (RMSD) as a guide should not be more than ±0.07. With SELCON3, the RMSD should not more than ±0.25.

Once basis set is selected, click estimate button and a 3D curve fit image (calculated spectra) is displayed as shown in the screenshot below.

Selecting components under graph choice on the right of the graph displayed, secondary structure contents are displayed as line graph for helix (combined alpha-helix and distorted helix), beta-strand (combined beta-strand and distorted beta-strand), turns and unordered.

Right clicking the mouse on the display panel will give you options to display the graph as 3D, 2D line graph or 2D bar chart as shown below.

All analyses are duplicated in excel workbook and an index page is created at the beginning of the work book listing all the names of the plot with their respective chart number as shown below. Clicking the appropriate chart number in the index sheet will take you to the chart number required in the excel workbook.

Generate Report
You can generate an experiment report by clicking "Generate Report". A 'Generate Word Report' window will appear listing figures that are available for incorporating into the word document.


Select the figures of interest and click "Generate Report" and save the file. An 'Experiment Word report generated' dialog will appear. Click OK and the saved Word report will be displayed on the screen.


The Word document will extract your beamtime proposal abstract from Usernet platform under the beamtime visit you have selected in the beginning. It will include experimental details, figures you have selected. You will only be required to insert your text discussion and conclusion together with future work. This report in its final version can be converted to pdf and submitted as a requirement of your 6 month post-beamtime report online in the Diamond Usernet.

User Documentation links
User documentation for CDApps: http://confluence.diamond.ac.uk/display/B23Tech/CD+Apps+documentation
Remote access to CDApps: https://remote.diamond.ac.uk/index.pl
Remote data transfer from Diamond: http://www.diamond.ac.uk/Home/ForUsers/academics/Documentation/Data-Transfer/TransferWindows/remote.html

User Documentation via CDApps
User documentation can also be accessed via CDApps by clicking the "User Documentation" button.


Once you click the 'User Documentation' button, a window containing links to the documentation, remote access and data transfer information can be viewed by clicking the appropriate button.

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