Residual Dipolar Coupling Experimental Setup

L. Kay's dipolar pulse sequences

general setup

fc180='n' except for checking 2D
f1180='y' since folding in 13C dimension - phase with -90, 180
on 500 sw=6600, sw1=1260, sw2=1700
may decrease sw2 depending on 15N dispersion
use Rance-Kay on last dimension
lp in 2nd and 3rd dimensions, then zero fill to 1024 (or 2048), 512, 64 (or 128)
coupling constant will be measured by splitting in 2nd dimension

Ha-Ca (hncoca_haca_dipolar)

Is not working correctly yet ?
X, Y, Z dimensions => H,C, N (after processing)
ni=82, ni2=32, at=.064, nt=4 -> 29 hours
can decrease ni2 to ni2=20
f_Iz='n','y' and array='f_Iz,phase2,phase'
separate fids as above
2 spectra give Co+J(CH)/2 and Co-J(CH)/2 in 2nd dimension
IPAP - see below for processing

Co-Ca (hnco_CoCa_ecosy_trosy_ydw)

X, Y, Z dimensions => H,C, N (after processing)
ni=82, ni2=32, at=.128, nt=4 -> 20.5 hours
can decrease ni2 to ni2=20
gives J of Co-Ca directly from separation in C dimension
H dimension gives HN-Ca but J was zero for unaligned (?)
coupled peaks in same spectrum - not IPAP
example nmrPipe script

N-Co (hnco_NCo_ecosy_trosy_ydw)

X, Y, Z dimensions => H, N, C (after processing)
must change order of axes during nmrPipe processing
ni=32, ni2=48, at=.128, nt=4 -> 12 hours
zero fill to 2048, 512, 128 (H, N, C)
Jf=4 (see sequence and publication)
2nd dimension is N and gives J of N-Co
15N + J(NH)/2 + (JF+1) * J(NCo)
1st dimension (proton) gives NH - J(NH)/2 + J(HnCo)
coupled peaks in same spectrum - not IPAP
example nmrPipe script

H-N (hnco_hn_coupling either trosy or no trosy)

X, Y, Z dimensions => H, N, C (after processing)
ni=32, ni2=42, at=.064, nt=4 (may decrease ni)
array='IP_flg,phase2,phase' (must be in this order)
sel_flg='y' for small proteins or small fields (trosy only)
2nd dimension gives J(NH) coupling
IPAP spectrum - coupled peaks are in separate spectra
separate fids using separate_3d.c, process each, and add and subtract (see below)
example nmrPipe script

H-N (hsqc_enhanced)2D

sw=10500, sw1=2200 on 800
ni=256, at=.064, nt=32 -> 15 hours
may decrease nt if concentrated sample
jxh = 95 (for HN)
IP_flg='n','y' and array='IP_flg,phase'
separate fids using separate.c, process each, and add and subtract (see below)

L. Kay's 3D ipap processing

general scheme - exact procedure depends on pulse sequence
maybe make a backup copy of the vnmr fid directory just in case

separate fid into the 2 interleaved data sets
  • 2D -> separate fid fid1 fid2 ni np
  • 3D -> separate_3d fid fid1 fid2 ni ni2 np
run varian conversion program ( on each fid
  • use Rance-Kay on z dimension (or y dimension for 2D)
  • 3D -> put results into directories data1 and data2
  • 2D -> put results into files test1.fid and test2.fid
process each data set as usual using nmrPipe, 1st checking the xy and xz planes
  • for y dimension, LP to 2x, then zero fill to 512
  • for z dimension, LP and ZF to 64 (or 128 if needed)
  • 3D -> put results into directories ft1 and ft2
  • 2D -> put results into files f1f3_1.dat and f1f3_2.dat
add and subtract the 2 transformed data sets (ft1 and ft2) using addNMR
  • 2D -> addNMR -in1 f1f3_1.dat -in2 f1f3_2.dat -out sum.dat -add
  • 2D -> addNMR -in1 f1f3_1.dat -in2 f1f3_2.dat -out dif.dat -sub
  • 3D -> addNMR -in1 ft1/test%03d.ft3 -in2 ft2/test%03d.ft3 -out sum/test%03d.ft3 -add
  • 3D -> addNMR -in1 ft1/test%03d.ft3 -in2 ft2/test%03d.ft3 -out dif/test%03d.ft3 -sub
convert into nmrView format
  • xyz2pipe -in sum/test%03d.ft3 -x -verb | pipe2xyz -nv -ov -out asum.nv
  • xyz2pipe -in dif/test%03d.ft3 -x -verb | pipe2xyz -nv -ov -out adif.nv
  • use similar commands for converting 2D spectra
name results asum.nv, adif.nv, usum.nv, and udif.nv for the aligned and unaligned sum and difference spectra
now open in nmrView and measure difference in C shifts using analysis scripts

BioPack Sequences


set up as usual for Nhsqc but set ipap flag to n,y
process and separte data as described in online manual

Chsqc (for measuring HaCa)

dm=nnn, dm2=nnn
ZZ=n, SE=y, CT=n, alphaC=y, COrefoc=y
coupled peaks are in same spectrum - not ipap

BioPack 2D sequences processed with nmrPipe

General method for interleaved IPAP spectra

separate fid into 2 fids using separate c program
  • separate fid fid1 fid2 ni np
translate data fomat using 'varian' program (
  • Nhsqc-IPAP will be complex in both dimentions.
  • Must do this for both fid1 and fid2
transform both translated fids using nmrPipe (ft2d script)
  • name resulting files f1f3_1.dat and f1f3_2.dat
  • for Nhsqc-IPAP, had to set 15N phase to 90, 0 for fid2
  • (used 0, 90 for earlier version of BioPack)
add and subtract fids using nmrPipe commands
  • addNMR -in1 f1f3_1.dat -in2 f1f3_2.dat -out sum.dat -add
  • addNMR -in1 f1f3_1.dat -in2 f1f3_2.dat -out dif.dat -sub
translate into nmrView format
  • nmrPipe -in sum.dat |pipe2xyz -nv -out sum.nv
  • nmrPipe -in dif.dat |pipe2xyz -nv -out dif.nv

gNtrosyS3 (BioPack)

First set phase=1,2 with small ni and check 2D
Then set phase=1,2,3,4 and use ni=512 with semitrosy='n'
nt need not be too large due to high sensitivity (try nt=8)
Separate, convert using Rance Kay for N15 and transform each spectrum with nmrPipe
may have to adjust both lp and rp phase parameters
Each spectrum will have one of the coupled partners.

See Dipoar Coupling analysis for more info