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How do you evaluate protein samples for the feasibility to solve 3D structure?

For example if you record a test HSQC set for a liquid state sample - what basic experimental parameters do you use and what S/N do you expect for a single peak?

What percentage of residues to you expect to be accounted for in the HSQC spectrum? Any other considerations?

Do you have a quantitative test?

asked Jul 20 '10 at 11:47

Evgeny%20Fadeev's gravatar image

Evgeny Fadeev

updated Jul 20 '10 at 20:13

2 Answers:
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According to my experience, I won't solely rely on the quality of HSQCs to test the feasibility of structure determeination. Of course, getting a high quality HSQC is a necessary condition. But, that is not the sufficient condition. I would rather record 3D-HNCO, 3D-HNCACB & 3D-CBCACONH. These experiments will tell you whether you have to go for "per deuterated sample" or not. In the past, in two cases, I got very good HSQCs. But, when I went for 3D experiments it was total failure. Those were 27 kDa & 28 kDa proteins. So, I had to go for perdeuteration and run TROSY based experiments to complete the backbone assignments. Downside with this is that you have to have a double labeled sample.

So, in short if you don't get a good HSQC - I won't go for double labeling. If I get a good HSQC, I'll keep my fingers crossed till I get good 3D-data using my double labeled sample.



answered Jul 21 '10 at 12:35

Ilango's gravatar image


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I suppose the following links from the NESG NMR WIKI (1)and (2) will be of help. Considering that we are using a high field cryoprobe equipped NMR spectrometer to record a high resolution 2D-[15N,1H] HSQC (tmax 15N ~ 110ms) spectrum, a signal to noise of 100:1 would mean rapid data acquisition methods (RD/GFT types) could be implemented, while 30:1 would mean conventional methods have to be employed.

The percentage of residues that appear in the HSQC (peak yield = 100*number of peaks observed/number of peaks expected) is a good measure of how well behaved the system is and the feasibility of structure determination could also be judged. A peak yield of >95% is good, while a challenging target might give around 80-85% (not very sure about this). If peak yield of more than 100% is obtained then either the possibility of the protein having more than one conformation is high, or it could be a dimer under the given set of conditions (could be verified by determining the rotational correlation time of the protein from relaxation measurements)

The peak dispersion would give a fair idea of whether the protein is unfolded or folded, if folded whether its a beta-rich or an alpha-rich protein. A good peak dispersion would mean that the protein is largely a beta-sheet rich protein, while a lower dispersion (with a good peak yield) would mean its a alpha-helix rich protein. A poor dispersion coupled with a poor peak yield implies unfolded proteins. I would like to add a note that, this is in reference to water soluble protein - the game with membrane proteins is quiet different.

Hope this helps!


answered Jul 21 '10 at 06:34

Bharathwaj's gravatar image


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