;HNCA[CB]-E.COSY for the Bruker AMX-600. ;Andy C. Wang and Ad Bax, March, 1995. ;3J(HnCb) from E.COSY splitting. ;Water is NOT saturated during experiment! Extremely low residual H2Oxy. ; 1. Flipped along z prior to gradients ; 2. Spin-locked along x during 1H decoupling ; 3. Uses sine-bell flip-back and Watergate prior to acquisition ;BMRB Pulse Sequence Accession Number: 32 ;_______________________________________________________________________ #include "bits.sg" #define CARBON #define NITROGEN #undef COADD_2D #define CARBON_LOOP 56 #define NITROGEN_LOOP 32 #define ITERATIONS 9 ;_______________________________________________________________________ ;PROTON pulses: ;p1 = 90 at hl1=3dB ;p2 = 900us 90 at hl2 = 45dB for Watergate pulse ;p4 = 1.7m half-gauss 90 at tp0 = 45dB for water-flip-back pulse ;p30 = 50us 90 at hl3 = 17dB for Dipsi-2 decoupling ;NITROGEN pulses: ;p3=nitrogen 90 at dbhi ;p31=nitrogen 90 at dbl0 ;CARBON pulses: ;carrier at 58 ppm ;p5=53.9u Ca 90 @ dl0=29 dB for null at 177 ppm ;p6=24.1u Ca 90 @ dl1=21 dB for null at 177 ppm ;p8=753u Ca 90-deg sinc @ dp1=46 dB ;p7=97.4u C' 90-deg offset sinc used as 180-deg @ dp0=30 dB ;p9=150u C' 90-deg offset sinc used as 180-deg @ dp2=33 dB ;p10=14.0u Ca pulse @ dl2=14 dB for composite 180 ;p11=14.0u Ca pulse @ dl2=14 dB for composite 180 ;p12=14.0u Ca pulse @ dl2=14 dB for composite 180 ;_______________________________________________________________________ ;Nitrogen constant time: d18=6.81m d10=d18-5.4m-p30 d19=d18 d20=d18 ;in10=in19=in20 ;in10=in20=in19=1/(4*sw) ;CARBON constant time: ;sw=1/(2*in0) ;_______________________________________________________________________ ;DELAYS: d2=5.6m-p9*2 d3=2.25m d4=d3-250u-p2 d5=5.5m-p9 d6=d5-p30-p9-4u p24=d4 ;d7 ca 100u = dw d8=aq d11=50m d12=8m d23=p3-p6 ;p3 > p6 d25=p3-p11*2.22 d26=p3-p1 d27=p9-p3 ;_______________________________________________________________________ ;f1 = hydrogen frequency on water ;f2 = Ca frequency 58 ppm ;f3 = nitrogen frequency ;_______________________________________________________________________ 100u CLEAR_BITS ;switch 2H decoupler off, switch everything off 100u LOCK_ON ;switch lock on ze 2 d11 dbo d12 3 d12*3 4 d12*2 5 d12*5 6 d12*6 7 10u dbo 10u do 10u to 100u LOCK_ON ;switch lock on d1 dlo 20m hl1 20u dbhi ;******* Start 90-degree ******************************** (p1 ph0):f1 d3 (d26 p1*2 ph0):f1 (p3*2 ph10):f3 d3 (p1 ph2):f1 50u LOCK_OFF 10u hl2 (p2 ph9):f1 ;flip water to +z GRAD( 10, POSITIV, 50.0 ) 3.3m (p3 ph3):f3 ;******* N15 evolution ******************************** 5.4m hl3 (p30 ph7):f1 ;put H2O on +x for spin-lock d10 cpdts dl6 ;(T+t2)/4 & dec. must be along x for H2O suppr. (p7*2 ph10):dp0 4u 2u d19 dl1 10u (d23 p6*2 ph6):f2 (p3*2 ph5):f3 2u 10u d20 dl6 (p7*2 ph10):dp0 4u d20 dl0 (p3 ph11):f3 20u (p5 ph4):f2 ;******* C-alpha evolution ******************************** 4u d0 dl6 ;t1/2 (d27 p3*2 ph1):f3 (p9*2 ph20):dp2 d0 4u dl6 ;******* back to N ******************************** (p8 ph17):dp1 (p3 ph10):f3 d5 (p9*2 ph20):dp2 d6 (p30):c8 4u dl2 (d25 p10 ph7 2u p11*2.44 ph0 2u p12 ph7):f2 (p3*2 ph10):f3 2u d2 dl6 (p9*2 ph10):dp2 2u to (p30 ph8):f1 ;put H2O back along +z 1m 4.4m hl1 ;******* back to Hn ******************************** (p3 ph0):f3 2u GRAD(70, NEGATIV, 20) 1.6m 5u tlo (p4 ph13):tp0 ;half-gauss 90 to flip water to +y 2u 2u thi 50u hl1 (p1 ph0):f1 GRAD(80, NEGATIV, 5) d4 hl2 (p2 ph14):f1 ;Watergate -x 90 pulse on water 2u 5u hl1 (p1*2 ph0):f1 2u 5u hl2 (p2 ph14):f1 (p3*2 ph10):f3 GRAD(90, NEGATIV, 5) 2u dblo (2u ph0) d7 dbl0 p24:e 3u adc d8 cpdb 10u do 10u to 10u dbo rcyc=2 ph31 d11 wr #0 if #0 zd ;_______________________________________________________________________ #ifdef CARBON d12 ip4 lo to 3 times 2 d12 id0 d12 ip31 d12 ip31 lo to 4 times CARBON_LOOP #endif ;_______________________________________________________________________ #ifdef NITROGEN d12 ip3 d12 rd0 lo to 5 times 2 d12 id10 d12 id19 d12 dd20 d12 ip31 d12 ip31 lo to 6 times NITROGEN_LOOP #endif ;______________________________________________________Co-add 2D spectra #ifdef COADD_2D d12 rf #0 d12 rp4 d12 rp31 d12 rd0 d12 CLEAR_BITS d12 ze lo to 7 times ITERATIONS #endif ;_______________________________________________________________________ 100u do 100u dbo 100u to 100u LOCK_ON ;switch lock on 100u CLEAR_BITS ;switch 2H decoupler off, switch everything off exit ;_______________________________________________________________________ ph0=0 ph1=1 3 ph2=1 ph3=0 ph4=0 0 2 2 ph5=0 0 0 0 1 1 1 1 ph6=0 ph7=1 ph8=3 ph9=(360)329 ;adjusted +x pulse for hl2 water-flip-back ph10=0 ph11=0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 ph13=(360)263 ;adjusted -x half-gauss water-flip-back pulse at tp0 ph14=(360)149 ;adjusted -x Watergate pulse at hl2 ph15=0 ph17=2 ;2 2 2 2 2 2 2 0 0 0 0 0 0 0 0 ph20=0 ph31=0 0 2 2 2 2 0 0 2 2 0 0 0 0 2 2 ;_______________________________________________________________________