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Dear NMR Wikiers ! I encountering problem with assignment of 12 kd protein ! i assigend already 50 percent of backbone using HNCA, HNCOCA CBCANH and CBCACONH , still some peaks are not quality of cbcanh and cbcaconh . how to assign rest of them using other experments ( HCCONH, HBHACONH ,CCCONH ) ? iam using sparky , vnmrj 700 mhz Thanking you anticipation |
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Try using 3D 15N edited NOESY and TOCSY, these are very complimentary to the triple resonance experiments. Also - in the case you have overlapped peaks - look at them in all three dimensions, that way you can resolve them better. Sparky allows switching the axes on the display plane easily. Command xr rotates the spectrum in 3D, also it helps to center view on a peak before rotations - select a peak and then type vc. Thanks alot for your reply - sri (May 11 '11 at 05:06) Noesy will help you make sequential assignments where triple resonance experiments can't. TOCSY will give HA's and maybe other protons on the chain. - Evgeny Fadeev (May 13 '11 at 12:44) |
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HCCONH, HBHACONH ,CCCONH are used for side chain assignment, not the backbone (but can help when there are ambiguity, because of discrimination of amino acid by side chain spin system). If you don't have enough pic in your CACBNH experiment (and CACBCONH), perhaps try more sensitive experiment set like HNCA/HN(CA)CO (CA and CO for aa i) and HN(CO)CA/HNCO (CA and CO for aa i-1), since you also have two carbon resonance to link amino acid together. This strategy is usually used for protein with a mass beyond 20kDa... Have you all pic in your 15N-HSQC? Have you an idea of secondary structure (by homology) ? What's the protein concentration ? I have enough pics , i had enough information about its structure ( x-ray cryatal structure reported ) . i am tring to solve by nmr and there are many proteins reported related same family .bcoz i need to dock with another protein . - sri (May 14 '11 at 12:44) Shall we guess any information from its secondary structure ? protein conc - 2mM - sri (May 14 '11 at 12:44) |
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SS can help you to anticipate NOE between Hn and other proton, so take together with a 3D 15N-noesy, that's help. Or simply, tell you about no structured region that can undergo ambiguity (and obviously, nHSQC tells you if protein are in good agreement with SS). If you are all CA/CB from CBCANH experiment, perhaps getting COi (HNcaCO) and COi-1(HNCO) can help a bit (3 instead 2 carbons to link aa) even if CO is a bad discriminator. Yes ! CO is bad discriminator by using HNCOCA/HNCO . I got very less information . CBCANH giving 60 percent pics are good .. Remaining are in abguity . So iam using CCCONH/HBHACONH experments to guess A.A residue - sri (May 15 '11 at 10:05) |
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Hi - have you tried using specific amino acid labelling? We use it regularly in our group, and have developed a fairly nice method that works really well. It's all published stuff - check out Jia, X. et al. J Biomol NMR 2009, 44, p59., and Xun-Cheng et al. J. Biomol NMR 2011, 50, p. 35. This uses the cell-free protein synthesis system. From five samples you can get unambiguous amino acid type assignments, which removes a lot of the ambiguities that you might encounter, even with small proteins like your one. Hope this helps, T |
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Why did you not use 3D HNCACB? I am using CBCANH EXPERIMENT . signal/noise ratio is very high and most of peaks no information about its back residue (i-1) and badly some peaks their is no information about its own residue (i) too . - sri (May 24 '11 at 02:30) |
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can some some pls educate me with HNCACB? im new to phd n have been on similar problem of 12 kda protien identification problem Manish, you should ask a separate question and try to explain what you would actually like to learn. It is not clear at all what is your question... - Evgeny Fadeev (Jun 17 '11 at 16:09) |
I submitted to pine NMR ( http://pine.nmrfam.wisc.edu/) It works for assignment very well and reverse label experiments helps me to remove out the ambiguity . - sri (Aug 25 '11 at 00:14)