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Dear NMR wiki ers

Iam working on protein family which can form dimers (total 20kd size ) using 25mM TRIS , 100 mM KCL Ph-6.8 , protein conc -2mM ( limitaion )

Iam always geting bad quality CBCANH experment on 700 vnmr cryob Could you please suggest me about expermental parameters or buffer conditions to get good quality of CBCANH experment

asked May 12 '11 at 11:58

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sri
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updated May 16 '11 at 10:19

Can you give us some acquisition parameter ? And have you test oligomerization (in other way, are you sure about dimer state ?) ? And How looks 15N-HSQC (all pic ? same shape ?) ? - Yoan Monneau (May 14 '11 at 12:21)

ralaxation delay- 0.6 sec water supression -251.2 hz 90 degree pulse width - 11.8u Sec complex points - 256 this protein already reported about its dimer state As pER SIZE EXCLUSION (fplc) RT at 70 showing dimer state cental region is little crowdy , 75 % same size , shape - sri (May 14 '11 at 15:23)

I deturated the protein randomly ( mass showing around 40 percent ). I have taken HNCACB instead of CBCANH . HNCACB is given good result for deuterated protein . - sri (Aug 25 '11 at 00:09)


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It's difficult to answer because we don't kwow what you mean by "bad quality" ! Described a bit your spectra can help somebody to answer...

Anyway, my impression is that recovery delay is a bit too short (typically 1s) and there are too much complex points (do you mean 256 complex points or 256 td1 point for 128 complex points ?) for aliphatic carbon evolution : you have to put a td1 with a certain limit because carbon evolution occur in a same time as polarization transfert to N (or CO in CBCACONH) that impose a limit (defined by coupling constant) of 3.6ms. Typically, 80 to 160 td1 points is enough to have a good resolution (with a SW of 80 ppm). Moreover, take account that decrease of S/N occur by splitting of 13C pic by homonuclear coupling if the evolution time is too long.

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answered May 16 '11 at 08:14

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Yoan Monneau
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updated May 16 '11 at 10:47

Thanks for your great help We always check the projections of f1f3,f2f3 and remain planes by keeping ralxation delay ( 0.5,0.6,0.7,0.8,0.9,1 sec array This gives best result we use to take that relaxation delay ( in my case 0.6 sec given best ) - sri (May 16 '11 at 10:15)

Bad quality means signal/noise ratio is high . 60 % shows its own i residue only . Rest of the cases no contour or signal about i-1 residue and some times its i residue in CBCANH experiment. - sri (May 16 '11 at 10:16)

yes, i mean 256 complex points . Next time I ll focus on that your given information - sri (May 16 '11 at 10:21)

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Usually the references can be found in the pulse sequence.

cbcaconh: ;using constant time in t2 ;S. Grzesiek & A. Bax, J. Biomol. NMR 3, 185-204 (1993) ;(D.R. Muhandiram & L.E. Kay, J. Magn. Reson. B 103, 203-216 (1994))

cbcanh: ;using constant time in t1 ;using constant time in t2 S. Grzesiek & A. Bax, J. Magn. Reson. 99, 201-207 (1992)

Also try to find an artivle by Dr. C Grisinger in Progress in NMR spectroscopy, 1993 or 1997, don't qiute remember. Will give you the reference later on.

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answered May 24 '11 at 09:45

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Wenjin Wu
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"Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients" M. Sattler, J. Schleucher and C. Griesinger Progress in Nuclear Magnetic Resonance Spectroscopy 34, 93-158 (1999) - Wenjin Wu (May 24 '11 at 17:10)

easier way: just compare the 2D H-CACB planes for CBCANH and CBCA(CO)NH (set ni of 13C to 96 or 128 that you would use for your 3D). Good luck. - Wenjin Wu (May 24 '11 at 17:12)

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Check out the last page (figure 46) of Bruker "3dmanual.pdf", it gave comparison of the 1st increment of some triple resonance experiments. You will also find the manual extremely useful. cheers,

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answered May 24 '11 at 17:17

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Wenjin Wu
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try CBCA(CO)NH.

CBCANH is not for a 20 kDa protein. It is with 13C-constant time evolution; while it removes CA-CB J-coupling, but with the price of sensitivity loss (more suitable for smaller proteins)

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answered May 22 '11 at 02:53

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Wenjin Wu
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Dear Wenjin Wu my protein is 10 kd , exist as dimer . could you please provide any reference for your suggestion i.e about CBCANH experiment . Regards sri - sri (May 24 '11 at 02:04)

"Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients" M. Sattler, J. Schleucher and C. Griesinger Progress in Nuclear Magnetic Resonance Spectroscopy 34, 93-158 (1999) - Wenjin Wu (May 24 '11 at 17:09)

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Do you get good sensitivity with the HNCA, HN(CO)CA, and HNCACB experiments? If not, you may need to deuterate the protein. In addition, we usually find that the CBCA(CO)NH experiment is more sensitive than the CBCANH experiment.

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answered Jun 01 '11 at 12:56

Eugene%20DeRose's gravatar image

Eugene DeRose
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HNCA AND HNCOCA SENSITIVITY IS HIGH . I GOT 100% PEAKS . CBCACONH SENSITIVITY IS ALSO GOOD . ONLY CBCANH EXPERIMENT ONLY GIVING TROUBLE . THANKS ALOT FOR YOUR SUGGESTION - sri (Jun 01 '11 at 22:00)

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