Supplementary Note 1: Bruker 2D long-range-H(N)CO-TROSY pulse sequence code. Semi-colons denote comments relating to symbols used to define the pulse scheme presented in Figure 2. The same color-coding as in Figure 2 is used here to highlight the various magnetization transfer, chemical shift evolution periods, etc. For the 3D version, the flag “#define NITROGEN” should be uncommented.
;2D or 3D long-range-HNCO-TROSY experiment for measuring h3JNC' correlations
;Typical pulse lengths are given for a 800 MHz spectrometer
#include "bits.incl"
; the file bits.incl contains C preprocessor definitions for channels, lock
; switching, etc (see below)
#define CARBONYL;uncomment for incrementation of carbonyl evolution
;#define NITROGEN;uncomment for incrementation of nitrogen evolution
;for the 3D version
;***** PROTON PULSES *****
;p1high-power 90 degree pulse at pl1
;p21ms 90 degree at pl2 (water flip-back pulse) adjust phcor14 and phcor18
;p32-3 ms 90 degree sinc1.0 pulse at sp0 (water flip-back pulse)
;***** CARBON PULSES *****
;p5carbonyl 90 degree sinc1.0 at sp1, set pl5=sp1; 110us @ 800 MHz
;p6carbonyl 180 degree sinc1.0 at sp1, set pl6=sp1; 220us @ 800 MHz
;p8Calpha 180 degree rect32 at sp2, set pl8=sp2; 17.8*2u @ 800 MHz (23.7*2u @ 600 MHz)
;***** NITROGEN PULSE *****
;p7high power nitrogen 90 degree at pl7
"p17=p7"
;***** GRADIENT PULSES *****
"p10=3.0m";G2 gradient pulse at gp0=+50% on +x
"p12=400u";G9 gradient pulse at gp0=+50% on +x
"p13=3.75m";G5 gradient pulse at gp0=+50% on +x
"p14=2.7m";G3 gradient pulse at gp0=+50% on +x
"p15=888u";G6 gradient pulse at gp0=+50% on +x
"p22=2.5m";G4 gradient pulse at gp2=+50% on y
"p16=10.0m";G1 and G7 gradient pulses at gp0=+50% on +x
"p18=700u";G8 gradient pulse at gp2=+50% on y
;***** DELAYS *****
;d16;16.5m for test, 66m for reference and cross experiment (T)
;d18;20u for test and cross, 16.5m for reference experiment (epsilon)
"d14=d16"
"d27=d18"
"d4=2.25m";H-N INEPT transfer delay (delta)
"d5=2.7m-p2-p12";N-H reverse-INEPT/TROSY transfer delay (delta’’)
"d11=50m";delay for disk I/O
"d12=10m";delay for disk I/O
"d17=d16-p14-25u";defines the first N-C’ INEPT period
"d19=d16-p14-p6-d18-25u";defines the second N-C’ INEPT period
"d24=p7*0.5-p1*0.5";delay to center 1H and 15N 90 degree pulses
"d25=p7-p8*0.5";delay to center 13CA and 15N 90 degree pulses
"d26=p7-p1";delay to center 1H and 15N 180 degree pulses
;***** Nitrogen chemical shift evolution *****
"d28=10u"
"d29=d14-d28-p8-p15-25u"
"d30=d14-d27-p6-p15-p3-1.565m";limitation for TD(15N): d30>0: l4 = d30/in30 –1
;in28 = in30 = 1/(2*sw(N)) dwell-time t2 = 1/(15N sweep-width in Hz) = in28+in30
;***** Carbonyl chemical shift evolution *****
"d15=in15*0.5-p5*0.5-p7-p8*0.5-4u";carbonyl incremental delay, sampling is delayed
;by 0.5*dw to reduce baseline offset
;in15 = 1/(2*sw(C’)) dwell-time t1 = 1/(13C’ sweep-width in Hz) = 2*in15
;***** Start of pulse sequence *****
1ze
21m unblank;turn deuterium lock ON
d11 do:C1
#ifdef CARBONYL
d12;delay for loop compensation
3d12*3
4d12*2
#endif
#ifdef NITROGEN
d12*7
5 d12*3
6 10u
#endif
10u pl7:N
10u pl5:C1
d1;recycle delay
1m blank;turn lock OFF (due to the use of gradients)
10u pl1:f1
1m
(p5:sp1 ph10):C1;90 degree purge pulse on 13C’
2u
p16:gp0;G1 gradient (suppress C’ Boltzmann magnetization)
10m
(p1 ph0);first 90 degree pulse on 1H
;***** H-N INEPT (time points a to b) *****
d4;INEPT transfer delay (delta)
(p7*2 ph10):N (d26 p1*2 ph4):H
d4;INEPT transfer delay (delta)
(p1 ph6):H
2u
10u pl2:f1
p2 ph18:r;low-power water flip-back pulse
2u
p10:gp0;G2 gradient pulse
2.5m pl1:f1
10u
1m
(p7 ph21):N;first 90 degree pulse on 15N
;***** N-C’ INEPT (time points b to d) *****
5u
p14:gp0;G3 gradient pulse
20u
d17 pl6:C1;first T period in N-C’ INEPT
(p7*2 ph27):N
20u pl7:N
d18 ;delay epsilon
(p6:sp1 ph20):C1
5u
p14:gp0;G3 gradient pulse
d19 pl5:C1;second (T-Epsilon) period in N-C’ INEPT
(p7 ph22):N
2u
p22:gp2;G4 gradient pulse
5m
(p5:sp1 ph23):C1;first 90 degree pulse on 13C’
;***** 13C’ chemical shift evolution (t1; time points d to e) *****
2u
d15 pl8:C1;first t1/2 evolution period
(p7*2 ph10):N;15N refocusing pulses
4u
(p8:sp2 ph25):C1;13CA refocusing pulse
2u
d15 pl5:C1;second t1/2 evolution period
;***** C’-N reverse-INEPT (time points e to g) *****
(p5:sp1 ph24):C1
4u
p13:gp0;G5 gradient pulse
5m;
;***** N15 evolution *****
if "l5==1" goto lab2; l5=1: ph3
(p17 ph13):N; l5=2: ph13
goto lab3
lab2, (p17 ph3):N
lab3, d28;t2/2 Nitrogen evolution
(p8:sp2 ph25):C1;13CA 180 degree refocusing pulse
5u
p15:gp0;G6 gradient pulse
20u pl7:N
d29 pl6:C1;first T period in C’-N reverse-INEPT
(p7*2 ph27):N
d27;delay epsilon
(p6:sp1 ph20):C1
20u
5u
p15:gp0;G6 gradient pulse
1.5m pl1:f1
44.5u;correction: (2/pi*p7)*2 - 6.5u
d30 ;Second (T-epsilon-t2/2) period
;in C’-N reverse-INEPT
5u
#endif
;********** TROSY part of the pulse sequence (time points g to h) **********
(p3:sp0 ph29):H;low-power water flip-back pulse
10u
10u
10u pl1:f1
5u
;***** N-H first reverse-INEPT *****
(p1 ph8):H
2u
p18:gp2;G8 gradient pulse
2.0m;delay delta
(p7*2 ph6):N (d26 p1*2 ph4):H
2u
p18:gp2;G8 gradient pulse
2.0m;delay delta
(p7 ph7):N (d24 p1 ph16):H
;***** N-H second reverse-INEPT *****
p12:gp0;G9 gradient pulse
d5;delay delta
5u
15u pl2:f1
(p2 ph14:r);low-power water flip-back pulse
2u
5u pl1:f1
(d26 p1*2 ph0) (p7*2 ph10):N
2u
5u pl2:f1
(p2 ph14:r);low-power water flip-back pulse
20u
p12:gp0;G9 gradient pulse
d5 pl31:C1;delay delta
(p7 ph17):N
(2u ph0)
;***** Acquisition (t2) *****
go=2 ph31 cpds3:C1;13C’ synchronized decoupling ON during t2
3u
10u do:C1;13C’ decoupling OFF
1m unblank;turn lock ON
d11 wr #0 if #0 zd
#ifdef CARBONYL
d12 ip23;incrementation of phase 23
lo to 3 times 2;loop for Real/Imaginary
d12 id15;incrementation of d15 for t1 evolution period
d12 ip31;incrementation of receiver phase
d12 ip31;incrementation of receiver phase
lo to 4 times l3;loop for l3 complex points in t1
d12 rd15;reset delay d15
d12 rp31;reset receiver phase
#endif
#ifdef NITROGEN
d12 ip16;p- and n-type N15 detection
d12 ip16
d12 ip7
d12 ip7
d12 ip29
d12 ip29
d12 iu5
lo to 5 times 2
d12 ru5
d12 id28;incrementation of d28 for t2 evolution period
d12 dd30;decrementation of d30 for t2 evolution period
lo to 6 times l4
#endif
d12 do:C1
d12 do:N
d12 LOCK_ON
exit
ph0=0
ph1=0
ph3= 1 1 2 2
ph13=1 1 0 0
ph4=1
ph6=3
ph7=3
ph8=1
ph10=0
ph14=2
ph16=2
ph17=2
ph18=2
ph20=0
ph21 = PHASE_4( 0 )
PHASE_4( 2 )
ph22=1
ph23=0 2
ph24=0
ph25=0
ph27=0
ph28=2
ph29=0
ph31=0 2 1 3 2 0 3 1
______
File bits.incl:
#define H f1
#define N f2
#define C1 f3
#define D f4
#define blank setnmr2|0 setnmr0|32 setnmr0|33 setnmr0|34
#define unblank setnmr2^0 setnmr0^32 setnmr0^33 setnmr0^34
#define PHASE_4( A ) A A A A
1