Supplementary Box 1: Bruker HNN-COSY Pulse Sequence Code

Supplementary Note 1: Bruker two-dimensional HNN-COSY pulse sequence code. Semi-colons denote comments relating to symbols used in the pulse scheme presented in Figure 3. The color coding highlights the various magnetization transfer steps and the 15N chemical shift evolution period of the pulse sequence code and matches the color shading presented in Figure 2. To record the two-dimensional HNN-COSY experiment on a 13C/15N-enriched sample, the flag “#define CARBON_LABEL” should be active, otherwise it should be commented out by a semi-colon as ";#define CARBON_LABEL".

File HNN-COSY:

;Two-dimensional HNN-COSY experiment for measuring h2JNN correlations

;bruker software version XWIN-NMR 3.5

#include "bits.incl"

; the file bits.incl contains C preprocessor definitions for channels, lock ; switching, etc (see below)

#define CARBON_LABEL

;****** PULSES ****** ;p1 high-power proton 90 degree pulse at pl1 ;p2 ~1.5 ms proton 90 at pl2 (water flip-back pulse) ; need to set phcor9, phcor14, phcor15 and phcor18 ;p7 high-power nitrogen 90 degree pulse at pl7

;p5 high-power carbon 90 pulse at pl5

;****** GRADIENT PULSES ****** "p10=2.5m" ;G1 gradient pulse length "p15=2.1m" ;G2 gradient pulse length "p16=2m" ;G3 gradient pulse length "p13=2m" ;G4 gradient pulse length "p18=200u" ;G5 gradient pulse length "p12=400u" ;G6 gradient pulse length "p14=4m*0.101322" ;G7 gradient pulse length

;****** NITROGEN CHEMICAL SHIFT EVOLUTION PERIOD ****** #ifdef CARBON_LABEL "d15=in15*0.5-p7*0.637-p5" ;Sampling is delayed by 0.5*dw #else "d15=in15*0.5-p7*0.637" ;Sampling is delayed by 0.5*dw #endif ;in15=1/(2sw) ;Increment 15 is equal to 1/15N sweep width in Hz

;****** DELAYS ****** "d4=2.25m" ;INEPT transfer delay (delta) "d11=50m" ;Delay for disk I/O "d12=10m" ;Delay for disk I/O "d13=25m" ;Delay for disk I/O "d26=p7-p1" ;Delay to center simultaneously applied 1H and 15N pulses

;2*d27 is the total length (2*delta) of 15N-15N COSY period ;d16 defines first COSY period, whereas d17 and d28 define second COSY period "d16=d27-10u-p15" "d17=d27-10u-p16" "d28=d27-64u-p13-p2"

;d24, d5 defines the delta delays in first and second reverse INEPT "d24=2.7m-p18" "d5=2.7m-p12" ;Delays d6, d7 define the epsilon delays before acquisition "d6=600u" "d7=d6-p14"

1 ;******** Start of pulse sequence ******** 1 ze 2 1m unblank d13 do:N d13 d12*2 3 d12*3 4 10u 10u pl7:N #ifdef CARBON_LABEL 10u pl5:C1 #endif d1 ;Recycle delay, usually set between 1 to 1.5 seconds 1m blank 10u pl1:H ;******** INEPT (time points a to b)******** (p1 ph0):H ;First 90 degree pulse on proton 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:H (p2 ph18:r):H ;Low-power water flip-back pulse 2u p10:gp0 ;G1 gradient pulse 2.5m pl1:H (p7 ph3):N ;First 90 degree pulse on nitrogen ;******** 15N-15N COSY transfer period (time points b to c) ******** 10u p15:gp1 ;G2 gradient pulse d16 ;First period in first COSY transfer period (p7*2 ph21):N 10u p15:gp1 ;G2 gradient pulse d16 ;Second period in first COSY transfer period (p7 ph22):N ;******** 15N Chemical shift evolution period (t1)(time points d to e) ******

d15 ;t1 evolution period #ifdef CARBON_LABEL (p5*2 ph20):C1 #endif d15 ;******** Second 15N-15N COSY interval (time points e to f) ******** (p7 ph23):N 10u p16:gp3 ;G3 gradient pulse d17 ;First period in second COSY transfer period (p7*2 ph10):N 10u p13:gp4 ;G4 gradient pulse with coherence selection d28 pl2:H ;Second period in second COSY transfer period (p2 ph9:r):H ;Low-power water flip-back pulse 4u 50u pl1:H ;******** TROSY part of the pulse sequence (time points f to g) ******** ;******** First reverse INEPT ******** (p1 ph8):H 4u p18:gp2 ;G5 gradient pulse d24 ;Epsilon delay period (p7*2 ph10):N (d26 p1*2 ph0):H 4u p18:gp2 ;G5 gradient pulse d24 ;Epsilon delay period (p7 ph7):N (p1 ph16):H ;******** Second reverse INEPT ******** 4u p12:gp0 ;G6 gradient pulse d5 ;Gamma(a) delay period (p7*2 ph10):N (d26 p1*2 ph0):H 4u p12:gp0 ;G6 gradient pulse d5 ;Gamma(a) delay period (p7 ph17):N d6

2 4u 93u (p1*2 ph0):H 4u p14:gp4 ;G7 gradient pulse with coherence selection d7 ;Gamma(d) delay period ;******** t2 (acquisition) period ******** go=2 ph31 1m unblank d11 wr #0 if #0 zd d12 ip3 ;Increment of phase 1 in Figure 1 d12 ip22 ;Increment of phase 2 in Figure 1 lo to 3 times 2 d12 id15 ;Increment for t1 evolution period d12 ip31 ;Increment of receiver phase d12 ip31 ;Increment of receiver phase lo to 4 times l3 d12 d12 do:N d12 unblank exit ph0=0 ph3=0 1 2 3 ph4=1 ph6=3 ph7=3 ph8=1 ph9=2 ph10=0 ph16=2 ph17=0 ph18=2 ph20=0 ph21=0 ph22=1 2 3 0 3 0 1 2 ph23=3 0 1 2 3 0 1 2 1 2 3 0 1 2 3 0 ph31=0 3 2 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