Principle of the CBCA(CO)NH determination

The magnetization is applied to the protons on C alpha and C beta.  There is chemical shift evolution such that signals appear on the carbon axis
for both C alpha, and C beta (if present).  C alpha is expected at a higher ppm value ( corresponding to the lesser electron density
at C alpha, but called a downfield shift).  Magnetization is transferred without evolution through the carbonyl to the n+1 amide.  After evolution on the N, it is transferred to
the amide proton and read out.  The N plane is a characteristic H x N HSQC-like spectrum, and the C plane correlates the n-1 C alpha and C beta
resonances with the amide signals.

Processing the CBCACONH N planes data.

The bruker strip:

#!/bin/csh

bruk2pipe -in ./ser -bad 0.0 -noaswap -DMX -decim 24 -dspfvs 12  \
  -xN              2048  -yN                82  \
  -xT              1024  -yT                41  \
  -xMODE            DQD  -yMODE  Echo-AntiEcho  \
  -xSW         7183.908  -ySW         1666.667  \
  -xOBS         600.083  -yOBS          60.813  \
  -xCAR           4.742  -yCAR         118.022  \
  -xLAB              1H  -yLAB             15N  \
  -ndim               2  -aq2D          States  \
  -out ./test.fid -verb -ov

sleep 5

Note the Echo-AntiEcho designation for yMODE

The nmrproc script:

#!/bin/csh

nmrPipe -in test.fid \
| nmrPipe -fn SOL                                          \
| nmrPipe  -fn SP -off 0.48 -end 0.98 -pow 3 -c 0.5        \
| nmrPipe  -fn ZF -size 2048                               \
| nmrPipe  -fn FT                                    \
| nmrPipe  -fn PS -p0 -33.0 -p1 0.0 -di -verb              \
| nmrPipe  -fn POLY -auto -ord 1                           \
| nmrPipe  -fn EXT -left -sw                               \
| nmrPipe  -fn TP                                          \
| nmrPipe  -fn SP -off 0.48 -end 0.98 -pow 2 -c 0.5         \
| nmrPipe  -fn ZF -size 512                               \
| nmrPipe  -fn FT  -neg                                  \
| nmrPipe  -fn PS -p0 0.0 -p1 0.0 -di -verb          \
| nmrPipe  -fn POLY -auto -ord 1                           \
| nmrPipe  -fn TP                                          \
   -ov -out test.dat

Note the -neg parameter on the second Fourier transform.  Without this the plot
comes out upside down on the N axis.

The Wisconsin version can NIH version looked about the same, although some peaks were brighter with one and some with the other.  By contrast to the HNCACB determination, signals with T2 < 80 , > 50 msec showed up at N=16.

C plane processing (from the pulse program):

;C13 Conversion/Processing
;Conversion, Dim=z, yMODE  Complex, aq2D  States
;Example processing...
;| nmrPipe  -fn SP -off 0.48 -end 0.98 -pow 2 -c 0.5         \
;| nmrPipe  -fn ZF -size 512                               \
;| nmrPipe  -fn FT                                     \
;| nmrPipe  -fn PS -p0 0.0 -p1 0.0 -di -verb          \
;| nmrPipe  -fn POLY -auto -ord 1                           \

3D processing scripts:

From Pete:

xyz2pipe   -in fid/test%03d.fid -x -verb
| nmrPipe  -fn SOL
...
|pipe2xyx -out data/testxy%03d.DAT -y -verb

xyz2pipe -in data/testxy%03d.DAT -z -verb
...
|pipe2xyz -out data/testxyz%03d.DAT -y -verb
 

Interpreting the strips:


The HNCA will give two C alpha signals for each amide signal.  These will be the n C alpha and
the n-1 C alpha.  (n-1 meaning the preceding residue in the sequence).  I think the n-1 signal is
expected to be weaker, but it may not be reliably so.  The n-1 signal is the one that also appears in
the CBCA(CO)NH determination correlated with this amide.  The C beta for the n-1 residue also appears in
CBCA(CO)NH strip correlated with amide n.
 

Issues:

I'm encountering a series of statements about why C alpha has a higher ppm value than C beta, which appear to contradict.   I need to sort this
out through some texts.  It is clear that C alpha has a higher ppm value.  It is clear that electron density of C alpha is lower than C beta due
to the carbonyl interaction.  So C alpha  experiences less shielding and hence a higher portion of the B0 field.  The frequency in Hz is therefore
higher since  the Larmour frequency is proportional to the field.

So one conflict is if you state the Lamour equation as freq =  gamma B0, then by definition you should say that the gamma value is different
between nuclei experiencing different chemical shifts.  But physically, it is more like that the gamma value is constant and the
effective field is altered (which Andy write Bloc).  What convention is used in general texts?

The second point of confusion is that upfield and downfield appear to be conventionally applied backwards to what actually happnes.  A shift
to higher ppm is called downfield, even though the nucleus is less shielded, and hence actually experiencing a greater field.  This then tends to
get even more confusing when people state downfield as "low field".  See how textbooks define/justify this convention.

Note: Andy made temporary space for me on /avance7001.