Processing HNCA data

Processing HNCA data.

Andy set up HNCA Wisconsin version and NIH version. I did N planes with just the NIH version (nhcafb.ref), then did a 3D determination with NS=24.
The N plane looked better than the cbcaconh N planes did, contrary to expectation.

Note on versions: Andy set up and checked N planes on ubiquitin for both Wisconsin and NIH versions of hnca on 8/3/2. Only the NIH version has been moved to ref. The datasets are in the ahinck directory on avance600 as hnca_wisc and hnca_nih. The pulse programs were hnca_ni.aph and hnca_wi.aph.

My processing scripts for N planes:

!/bin/csh

bruk2pipe -in ./ser -bad 0.0 -noaswap -DMX -decim 24 -dspfvs 12 \
-xN              2048 -yN                90 \
-xT              1024 -yT                45 \
-xMODE            DQD -yMODE        Complex \
-xSW         7183.908 -ySW         1666.666 \
-xOBS         600.083 -yOBS          60.813 \
-xCAR           4.742 -yCAR         118.034 \
-xLAB              1H -yLAB             15N \
-ndim               2 -aq2D          States \
-out ./test.fid -verb -ov

sleep 5

I originally processed this plane iwth N SW = 2222.222, which I must have been asleep to get somehow. According to the pulse program, SW should have been 1/4*IN21 which is 1666.666. All this mistake does is change the N scale. Sure enough, 1666.666 (even though the bruker script doesn't cough it up) is the number that gives the correct coordinates for the known HSQC pattern.

#!/bin/csh

#
# Basic 2D Phase-Sensitive Processing:
#   Cosine-Bells are used in both dimensions.
#   Use of "ZF -auto" doubles size, then rounds to power of 2.
#   Use of "FT -auto" chooses correct Transform mode.
#   Imaginaries are deleted with "-di" in each dimension.
#   Phase corrections should be inserted by hand.
# Modified for nhca_fb N plane processing.

nmrPipe -in test.fid \
| nmrPipe -fn SOL    \
| nmrPipe -fn SP -off 0.50 -end 1.00 -pow 1 -c 0.5    \
| nmrPipe -fn ZF -auto                               \
| nmrPipe -fn FT -auto                           \
| nmrPipe -fn PS -p0 56.8 -p1 8.6 -di -verb         \
| nmrPipe -fn EXT -left -sw                                    \
| nmrPipe   -fn POLY -ord 2 \
| nmrPipe    -fn TP      \
| nmrPipe -fn SP -off 0.45 -end 0.98 -pow 2 -c 0.5    \
| nmrPipe -fn ZF -size 128                           \
| nmrPipe -fn FT -auto                               \
| nmrPipe -fn PS -p0 0.0 -p1 0.00 -di -verb         \
| nmrPipe -fn POLY -auto -ord 1       \
| nmrPipe -fn TP                           \
-ov -out test.ft2

3D processing:

Note: In the NIH HNCA program, C is collected on the Y axis, and N on the Z axis.
Also, there is about a 10 degree phase correction on the 2nd dimension probably due
to some delay time being not exactly right. Just judge it by nmrDraw and make it
in the usual way in the script.

Andy looked at my test C plane and noted the row of spots on the top were glycines.
There are more than expected, which may be the equilibrium with denatured forms.

3D bruk2pipe script:

#!/bin/csh

bruk2pipe -in ./ser -bad 0.0 -noaswap -DMX -decim 24 -dspfvs 12 \
-xN              2048 -yN               130 -zN                90 \
-xT              1024 -yT                65 -zT                45 \
-xMODE            DQD -yMODE        Complex -zMODE        Complex \
-xSW         7183.908 -ySW         4098.361 -zSW         1666.666 \
-xOBS         600.083 -yOBS         150.898 -zOBS          60.813 \
-xCAR           4.742 -yCAR          56.044 -zCAR         118.034 \
-xLAB              1H -yLAB             13C -zLAB             15N \
-ndim               3 -aq2D          States                         \
-out ./fid/test%03d.fid -verb -ov

My testC processing script.

#!/bin/csh
#This version for C plane of 3D hncac data.
#Note: since C is on Y and N on Z, you do the test
# script for test001 to make a C plane.
# Expect two positive spots aligning with each
# 1H signal.

nmrPipe -in ./fid/test001.fid \
| nmrPipe -fn SOL                                          \
| nmrPipe -fn SP -off 0.46 -end 0.98 -pow 1 -c 0.5        \
| nmrPipe -fn ZF -size 2048                               \
| nmrPipe -fn FT                                         \
| nmrPipe -fn PS -p0 55.0 -p1 0.0 -di -verb              \
| nmrPipe -fn POLY -auto -ord 1                           \
| nmrPipe -fn EXT -left -sw                               \
| nmrPipe -fn TP                                          \
| nmrPipe -fn SP -off 0.45 -end 0.98 -pow 2 -c 1.0         \
| nmrPipe -fn ZF -size 128                               \
| nmrPipe -fn FT                                        \
| nmrPipe -fn PS -p0 -169.8 -p1 360.0 -di -verb          \
| nmrPipe -fn POLY -auto -ord 1                           \
| nmrPipe -fn TP                                          \
   -ov -out test.dat

The overall 3D scheme:
Input HCN as -x
After processing H, fn TP to make CHN
After processing C, output -y to remake HCN
The first testxy file will be a readable HC plane
input as -z to make NHC
process N
Then fn ZTP to make CHN,
then output
as -y to make HCN.

The 3d processing script:
#!/bin/csh
#This version for 3D hncac data.
#Note: C is on Y and N on Z

xyz2pipe -in ./fid/test%03d.fid -x -verb           \
| nmrPipe -fn SOL                                          \
| nmrPipe -fn SP -off 0.46 -end 0.98 -pow 1 -c 0.5        \
| nmrPipe -fn ZF -size 2048                               \
| nmrPipe -fn FT                                         \
| nmrPipe -fn PS -p0 55.0 -p1 0.0 -di               \
| nmrPipe -fn POLY -auto -ord 1                           \
| nmrPipe -fn EXT -left -sw                               \
| nmrPipe -fn TP                                          \
| nmrPipe -fn SP -off 0.45 -end 0.98 -pow 2 -c 1.0         \
| nmrPipe -fn ZF -size 128                               \
| nmrPipe -fn FT                                        \
| nmrPipe -fn PS -p0 -169.8 -p1 360.0 -di           \
| nmrPipe -fn POLY -auto -ord 1                           \
| pipe2xyz -ov -out data/testxy%03d.dat -y

Phase problem:

It was observed that phase came out wrong in the C dimension after processing the 3rd (N) dimension. Re-examination of the testxy files (HC planes) shows that the phase offset varied through the series in a linear fashion from 10.2 in test001 to 82 in test090. I suspect some problem in the pulse program either due to Andy's changes (in particular he was doing something to the lock hold), or to some peculiarity in porting the code to the Bruker system, or to something bizzare related to the probe problem. Andy prefers to think that there was something changing during the experiment. The plan is to fix the probe, and then re-examine this problem using ubiquitin. Meanwhile, to save the data, will make a script to process each fid to a testxy file with a separate phase calculated as -180 + (9.39 + n* 0.81)

Here's the perl script to make the processing script:

# Into process.com put 90 lines of the form
# auto2d.com ./fid/testn.fid y ./data/testxyn.dat
# where n is 3 digits running from 1 to 90
# and y = -180 + (9.38 + 0.81 * n)

# Execute by perl mkscript.pl

open (OUT, '>process.com') or die "Can not open process.com\n";
print OUT ('#!/bin/csh'."\n");
for ($n=1;$n<91;$n++)
{$y = 9.38 + $n * 0.81 -180;
printf OUT ("auto2d.com ./fid/test%03d.fid %6.2f ./data/testxy%03d.dat\n",$n,$y,$n);
}
close (OUT);

The second part of the processing was carried out by:

#!/bin/csh
#This script does the 2nd part of the hnca 3d processing.
# It expects the testxy files to have been put out in HCN order.

NOTE: Expect a long delay after the last -verb echo before this finishes.

Issues:
Type pepdata <input file of seq> <output file>
to get amino acid compositions.

Where does this phase problem come from? What about the winsconsin version

Andy thinks that the cbcaconh should be repeated, maybe with the nih version, because it's expected to
come out stronger than this experiment.