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 ;#define NITROGEN ;uncomment for incrementation of carbonyl evolution ;uncomment for incrementation of nitrogen evolution ;for the 3D version ;***** PROTON PULSES ***** ;p1 high-power 90 degree pulse at pl1 ;p2 1ms 90 degree at pl2 (water flip-back pulse) adjust phcor14 and phcor18 ;p3 2-3 ms 90 degree sinc1.0 pulse at sp0 (water flip-back pulse) ;***** CARBON PULSES ***** ;p5 carbonyl 90 degree sinc1.0 at sp1, set pl5=sp1; 110us @ 800 MHz ;p6 carbonyl 180 degree sinc1.0 at sp1, set pl6=sp1; 220us @ 800 MHz ;p8 Calpha 180 degree rect32 at sp2, set pl8=sp2; 17.8*2u @ 800 MHz (23.7*2u @ 600 MHz) ;***** NITROGEN PULSE ***** ;p7 high 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" "d5=2.7m-p2-p12" ;H-N INEPT transfer delay (delta) ;N-H reverse-INEPT/TROSY transfer delay (delta’’) "d11=50m" "d12=10m" ;delay for disk I/O ;delay for disk I/O "d17=d16-p14-25u" ;defines the first N-C’ INEPT period 1 "d19=d16-p14-p6-d18-25u" "d24=p7*0.5-p1*0.5" "d25=p7-p8*0.5" "d26=p7-p1" ;defines the second N-C’ INEPT period ;delay to center 1H and 15N 90 degree pulses ;delay to center 13CA and 15N 90 degree pulses ;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 ***** 1 ze 2 1m unblank ;turn deuterium lock ON d11 do:C1 #ifdef CARBONYL d12 ;delay for loop compensation 3 d12*3 4 d12*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 2 (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 3 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 4 d12 ru5 d12 id28 d12 dd30 lo to 6 times l4 #endif ;incrementation of d28 for t2 evolution period ;decrementation of d30 for t2 evolution period 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 #define #define #define H f1 N f2 C1 f3 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 5