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Molecular weight analysis
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
.. _raw_mw:
This tutorial covers how to use RAW for molecular weight analysis. This is not a
tutorial on basic principles and best practices for molecular weight analysis.
For that, please see the :ref:`SAXS tutorial <saxs_mw>`.
RAW provides four methods of molecular weight analysis:
* Referencing I(0) to that of a known standard
* From the volume of correlation using the method of Rambo and Tainer
* From the adjusted Porod volume using the method of Fisher et al.
* From the value of I(0) on an absolute scale.
If ATSAS is installed, RAW also provides an additional two methods of MW
calculation using the ATSAS tools, for a total of six different methods:
* From classification by machine learning (ATSAS datclass/Shape&Size)
* From a Bayesian estimation based on concentration independent methods (ATSAS
datmw bayes)
If you use these MW methods in RAW, in addition to the RAW paper please cite
these papers:
* Volume of correlation: Rambo, R.P. & Tainer, J.A. Nature (2013). 496,
477-481. DOI: `10.1038/nature12070 <https://doi.org/10.1038/nature12070>`_
* Corrected Porod volume: V. Piiadov, E. Ares de Araujo, M. Oliveira Neto, A. F.
Craievich, and I. Polikarpov. Protein Science (2019). 28(2), 454-473.
DOI: `10.1002/pro.3528 <https://doi.org/10.1002/pro.3528>`_
* Bayesian inference (ATSAS): Hajizadeh, N. R., Franke, D., Jeffries, C.
M. & Svergun, D. I. (2018). Sci. Rep. 8, 7204.
DOI: `10.1038/s41598-018-25355-2 <https://doi.org/10.1038/s41598-018-25355-2>`_
* Shape&Size (ATSAS): Franke, D., Jeffries, C. M. & Svergun, D. I. (2018).
Biophys. J. 114, 2485–2492.
DOI: `10.1016/j.bpj.2018.04.018 <https://doi.org/10.1016/j.bpj.2018.04.018>`_
A video version of this tutorial is available:
.. raw:: html
<style>.embed-container { position: relative; padding-bottom: 56.25%; height: 0; overflow: hidden; max-width: 100%; } .embed-container iframe, .embed-container object, .embed-container embed { position: absolute; top: 0; left: 0; width: 100%; height: 100%; }</style><div class='embed-container'><iframe src='https://www.youtube.com/embed/qYWzhBdnsRY' frameborder='0' allowfullscreen></iframe></div>
The written version of the tutorial follows.
#. In RAW, right click on the subtracted GI scattering profile in the Profiles panel
and select “Molecular weight.” Alternatively click on the "Mol. Weight" button
at the bottom of the Profiles panel.
|mw_open_png|
#. At the top of the panel are the results of the Guinier fit. All methods
require a good Guinier fit, so you can use that button to redo the fit if
necessary. In the lower part of the panel, the results of the estimates
for MW are shown.
* *Note:* Neither the I(0) Ref. MW panel nor the Abs. MW panel should be reporting a MW.
* *Note:* If you have ATSAS installed and accessible to RAW, you should see
six panels with MW, as in the image below. If you don't have ATSAS
installed you will see just four panels, the rightmost panel in each
row will be missing.
* *Tip:* To learn more about any of the methods, click on the "More Info"
button.
|mw_png|
#. In either concentration box, enter the sample concentration of 0.47 mg/ml. Notice that you
now get results from all methods of MW calculation.
* *Question:* The expected MW value for GI is 172 kDa. How do your results compare?
#. Click on the “Show Details” button for the Vc MW panel. You should see a graph, which shows
the integrated area of :math:`qI(q)` vs. *q*\ . For this method to be accurate,
this value needs to converge at high *q*\ .
|mw_vc_png|
#. Click on the "Show Details" for the Vp MW panel. You'll notice that both Vp MW
and Vc MW have a "cutoff" selection. At higher q you can start to get
scattering from flexibility or intra-molecular features that may reduce the
reliability of the MW estimate. RAW automatically cuts off the scattering profile
used based on the size of the object. You can change the cutoff if you need to.
|mw_cutoff_png|
#. Click the “OK” button to save your analysis.
* *Note:* The “Cancel” button discards the analysis.
* *Tip:* After clicking "OK" you can now click on the GI profile in the
Profiles control panel and see the MW you just found in the Info panel.
#. Repeat the MW analysis for the lysozyme sample, which had a concentration of 4.27 mg/ml.
The expected MW of lysozyme is 14.3 kDa.
* *Question:* Does the Vc method work for the lysozyme data?
.. |mw_open_png| image:: images/mw_open.png
:width: 400 px
:target: ../_images/mw_open.png
.. |mw_png| image:: images/mw.png
:target: ../_images/mw.png
.. |mw_vc_png| image:: images/mw_vc.png
:width: 300 px
:target: ../_images/mw_vc.png
.. |mw_cutoff_png| image:: images/mw_cutoff.png
:width: 600 px
:target: ../_images/mw_cutoff.png
|
