# 1 "/u/exp/stan/nmr/lists/pp/ko_cryo_HCCCONNH3d.3" ;ko_hccconnh << Yutaka Ito Ver.13/7/1999 ;FLOPSY-16 version ;for Avance consoles. ;Constant time 3D H(CCCO)NNH. ;S.Grzesiek and A.Bax, J.Magn.Reson.Ser B,1993,101,114-119. ;R.T.Clowes et al., J.Biomol.NMR,1993,3,349-354. ;D.Nietlispach et al., J.Am.Chem.Soc.1996,118,407-415. ;with water-flip-back pulses and Watergate for the 15N->1H INEPT. ;Phase-sensitive mode using States(F1) and States-TPPI(F2). ; ;F1(1H)-ch: ;p1:1H 90deg @pl1 [~10.0us@-5.0dB] ;p5:1H 90deg @pl5 [1.0ms@29dB] (phcor7,8,9) ;p7:1H 90deg @pl7 [~55us@7.5dB] ;pcpd1:1H CPD @pl7 [~55us@7.5dB] ;pl1:1H hard pulse [p1] ;pl5:1H wfb pulse [p5] ;pl7:1H CPD pulse [p7,pcpd1] ; ; ; ;F3(13CA/CB)-ch: ;p13:CAB 90deg @pl2,sp0 [48.0u@8.0dB] ;p26:CAB 180deg @pl11,sp8 [21*2@3.2dB] ;p8:Cmix 90deg @pl15 [34.4u@5.0dB] ;p15:CA 180deg rSNOB(i_rsnob_100) @sp5 ;p23:CAB 180deg G3.256 @sp7 ;pl2:CAB 90deg ~49u@10.18dB ;pl11:CAB 180deg 21u*2@3.2dB ;pl15:Cmix ~35u@7.1dB ;sp0:CAB 90deg rect ~48u@8.0dB ;sp5:CA 180deg rSNOB 367u@8.2dB ;sp7:CAB 180deg G3.256 256u@-0.9dB. ;sp8:CAB 180deg rect @pl11 ;l1:mixing cycle ; (p8*188.448*l1) ; ;F3(13C')-ch: ;p14:CO 90deg @pl8,sp3 ;p24:CO 180deg SEDUCE1 @sp2,4 ;pl8:CO 90deg ~53u@10.5dB ;sp3:CO 90deg rect ~53u@10.5dB ;sp4:CO 180deg SEDUCE1 192u@9.5dB ;sp2:CO 180deg SEDUCE1 192u@9.5dB ; ; ;F2(15N)-ch: ;p21:90deg @pl3 ;pcpd3:90deg @pl13 ;pl3:~49u @0dB ;pl13:~250u @14.5dB(for CPD). ;in30: =in10 (d30>in30*l4) ;Gradients (total: 7). ; p25-p25 : echo. ; p19 ; p16 : spoil. ; p17 : spoil. ; p30-p30 : echo (water-gate). # 1 "/u/exp/stan/nmr/lists/pp/Avance.incl" 1 ;Avance.incl ; ;avance-version (03/02/17) ;$Id: pulseprogram,v 1.1 2021/02/10 23:29:09 bmrbsvc Exp $ # 67 "/u/exp/stan/nmr/lists/pp/ko_cryo_HCCCONNH3d.3" 2 # 1 "/u/exp/stan/nmr/lists/pp/Grad.incl" 1 ;Grad.incl - include file for Gradient Spectroscopy ; ;avance-version (02/05/31) define list EA= ;$Id: pulseprogram,v 1.1 2021/02/10 23:29:09 bmrbsvc Exp $ # 68 "/u/exp/stan/nmr/lists/pp/ko_cryo_HCCCONNH3d.3" 2 ;defined parameters. ;gradient pulses. ;p25:400u gp1 : echo ;p19:2.0m gp2 : homospoil ;p27:1.5m gp3 : homospoil ;p28:2.0m gp4 : homospoil ;p29:3.0m gp5 : homospoil ;p30:800u gp6 : echo ;for adjusting centre of 180 pulses. "d11=p23*0.5-p1" ; 13CA/CB_180*0.5 - 1H_90. "d12=p24*0.5-p21" ; 13CO_180*0.5 - 15N_90. "d13=p21-p1" ; 15N_90 - 1H_90. ;for t1(1H) evolition and 1H->13C INEPT. "d0=4u" "d3=1.5m-p25-100u" "d8=8u+p26" "d4=1.05m-p23*0.5" ;for 13CA->13C' transfer. "d14=3.24m+16u" "d15=3.24m" ;for 13CO->15N transfer. "d16=4.4m-p7-9.5u" "d17=4.4m-p7-33.5u" ;for t2(15N) evolution. "d7=p7" "d10=0.6m-9.5u-p7-p15*0.5" "d30=6m-9.5u-p7-p15*0.5" ;for 15N->1H transfer and acquisition. "d6=2.25m-p30-124u-p5" "l3=td1/2" "l4=td2/2" "in30=in10" "spoff0=0" "spoff2=0" "spoff3=0" "spoff4=bf2*((cnst21-cnst23)/1000000)" "spoff5=bf2*((cnst22-cnst21)/1000000)" "spoff7=0" "spoff8=0" ;pulse sequence. 30m setnmr2^0 setnmr0^34^32^33 ; gradient-blanking on. ;---------- 20u do:f2 20u do:f3 ze 1 70m do:f3 ; 15N CPD stop. 2 30m 3 30m 4 30m ; relaxation delay = d1 + 160ms. ;---------- INEPT 1H->13C. 5 d1 fq=cnst23(bf ppm):f2 20u pl1:f1 ; 1H power -> pl1. 50u setnmr2|0 setnmr0|34|32|33 20u pl3:f3 ; 15N power -> pl3. ;---------- (p1 ph0):f1 ;========== 1st gradient pulse (echo). 5u p25:gp1 95u ;========== d0 d3 pl11:f2 ; 13CA/B power -> pl11. (p26:sp8 ph12):f2 d0 (p1*2.0 ph1):f1 ;========== 2nd gradient pulse (echo). 5u p25:gp1 95u ;========== d8 d3 pl2:f2 ; 13CA/B power -> pl2. (p1 ph2):f1 ;---------- 13CB->13CA transfer. (p13:sp0 ph11):f2 d4 (d11 p1*2.0 ph1):f1 (p23:sp7 ph14):f2 d4 (p13:sp0 ph13):f2 (p1 ph2 4u p1 ph5):f1 ; water -> Iz ;---------- 13C isotropic mixing DIPSI-3. 4u ;========== 5u p19:gp2 95u ;========== 100u pl15:f2 ; 13C power -> pl15. ;---------- ;R 9 (p8*0.511 ph15):f2 (p8*1.067 ph16):f2 (p8*1.822 ph17):f2 (p8*1.767 ph18):f2 (p8*1.444 ph19):f2 (p8*1.767 ph18):f2 (p8*1.822 ph17):f2 (p8*1.067 ph16):f2 (p8*0.511 ph15):f2 ;Rbar (p8*0.511 ph25):f2 (p8*1.067 ph26):f2 (p8*1.822 ph27):f2 (p8*1.767 ph28):f2 (p8*1.444 ph29):f2 (p8*1.767 ph28):f2 (p8*1.822 ph27):f2 (p8*1.067 ph26):f2 (p8*0.511 ph25):f2 ;Rbar (p8*0.511 ph25):f2 (p8*1.067 ph26):f2 (p8*1.822 ph27):f2 (p8*1.767 ph28):f2 (p8*1.444 ph29):f2 (p8*1.767 ph28):f2 (p8*1.822 ph27):f2 (p8*1.067 ph26):f2 (p8*0.511 ph25):f2 ;R (p8*0.511 ph15):f2 (p8*1.067 ph16):f2 (p8*1.822 ph17):f2 (p8*1.767 ph18):f2 (p8*1.444 ph19):f2 (p8*1.767 ph18):f2 (p8*1.822 ph17):f2 (p8*1.067 ph16):f2 (p8*0.511 ph15):f2 ;R (p8*0.511 ph15):f2 (p8*1.067 ph16):f2 (p8*1.822 ph17):f2 (p8*1.767 ph18):f2 (p8*1.444 ph19):f2 (p8*1.767 ph18):f2 (p8*1.822 ph17):f2 (p8*1.067 ph16):f2 (p8*0.511 ph15):f2 ;R (p8*0.511 ph15):f2 (p8*1.067 ph16):f2 (p8*1.822 ph17):f2 (p8*1.767 ph18):f2 (p8*1.444 ph19):f2 (p8*1.767 ph18):f2 (p8*1.822 ph17):f2 (p8*1.067 ph16):f2 (p8*0.511 ph15):f2 ;Rbar (p8*0.511 ph25):f2 (p8*1.067 ph26):f2 (p8*1.822 ph27):f2 (p8*1.767 ph28):f2 (p8*1.444 ph29):f2 (p8*1.767 ph28):f2 (p8*1.822 ph27):f2 (p8*1.067 ph26):f2 (p8*0.511 ph25):f2 ;Rbar (p8*0.511 ph25):f2 (p8*1.067 ph26):f2 (p8*1.822 ph27):f2 (p8*1.767 ph28):f2 (p8*1.444 ph29):f2 (p8*1.767 ph28):f2 (p8*1.822 ph27):f2 (p8*1.067 ph26):f2 (p8*0.511 ph25):f2 ;Rbar (p8*0.511 ph25):f2 (p8*1.067 ph26):f2 (p8*1.822 ph27):f2 (p8*1.767 ph28):f2 (p8*1.444 ph29):f2 (p8*1.767 ph28):f2 (p8*1.822 ph27):f2 (p8*1.067 ph26):f2 (p8*0.511 ph25):f2 ;R (p8*0.511 ph15):f2 (p8*1.067 ph16):f2 (p8*1.822 ph17):f2 (p8*1.767 ph18):f2 (p8*1.444 ph19):f2 (p8*1.767 ph18):f2 (p8*1.822 ph17):f2 (p8*1.067 ph16):f2 (p8*0.511 ph15):f2 ;R (p8*0.511 ph15):f2 (p8*1.067 ph16):f2 (p8*1.822 ph17):f2 (p8*1.767 ph18):f2 (p8*1.444 ph19):f2 (p8*1.767 ph18):f2 (p8*1.822 ph17):f2 (p8*1.067 ph16):f2 (p8*0.511 ph15):f2 ;Rbar (p8*0.511 ph25):f2 (p8*1.067 ph26):f2 (p8*1.822 ph27):f2 (p8*1.767 ph28):f2 (p8*1.444 ph29):f2 (p8*1.767 ph28):f2 (p8*1.822 ph27):f2 (p8*1.067 ph26):f2 (p8*0.511 ph25):f2 ;Rbar (p8*0.511 ph25):f2 (p8*1.067 ph26):f2 (p8*1.822 ph27):f2 (p8*1.767 ph28):f2 (p8*1.444 ph29):f2 (p8*1.767 ph28):f2 (p8*1.822 ph27):f2 (p8*1.067 ph26):f2 (p8*0.511 ph25):f2 ;Rbar (p8*0.511 ph25):f2 (p8*1.067 ph26):f2 (p8*1.822 ph27):f2 (p8*1.767 ph28):f2 (p8*1.444 ph29):f2 (p8*1.767 ph28):f2 (p8*1.822 ph27):f2 (p8*1.067 ph26):f2 (p8*0.511 ph25):f2 ;R (p8*0.511 ph15):f2 (p8*1.067 ph16):f2 (p8*1.822 ph17):f2 (p8*1.767 ph18):f2 (p8*1.444 ph19):f2 (p8*1.767 ph18):f2 (p8*1.822 ph17):f2 (p8*1.067 ph16):f2 (p8*0.511 ph15):f2 ;R (p8*0.511 ph15):f2 (p8*1.067 ph16):f2 (p8*1.822 ph17):f2 (p8*1.767 ph18):f2 (p8*1.444 ph19):f2 (p8*1.767 ph18):f2 (p8*1.822 ph17):f2 (p8*1.067 ph16):f2 (p8*0.511 ph15):f2 4u lo to 9 times l1 ; Mixing time: p8*188.448*l1 4u 20u pl2:f2 ; 13C power -> pl2. ;---------- 13CA -> 13C' INEPT. (p13:sp0 ph13):f2 4u 20u pl7:f1 ; 1H power -> pl7, for CPD. (p7 ph1):f1 5.5u 4u cpds1:f1 ; start 1H CPD. (p24:sp4 ph10):f2 ; cancel Bloch-Siegert effect. d15 (p23:sp7 ph12):f2 ; sel 180 pulse for 13CA/B. 4u (p24:sp4 ph10):f2 ; sel 180 pulse for 13CO. 4u d14 pl2:f2 ; 13C power -> pl2. 4u do:f1 ; stop 1H CPD. 5.5u (p7 ph4):f1 (p13:sp0 ph10):f2 ;========== 3rd gradient pulse (homo-spoil). 5u p27:gp3 95u ;========== 30u fq=cnst21(bf ppm):f2 200u pl8:f2 ; 13CO power -> pl8. ;---------- INEPT 13C'-15N. (p14:sp3 ph12):f2 (p7 ph1):f1 5.5u 4u cpds1:f1 ; start 1H CPD. d16 (p15:sp5 ph10):f2 ; sel 180 pulse for 13CA (off-resonance). 8.0m (p24:sp2 ph10):f2 (d12 p21*2.0 ph20):f3 d17 8.0m (p15:sp5 ph10):f2 ;cancel Bloch-Siegert Effect. 4u 20u pl8:f2 ; 13CO power -> pl8. 4u do:f1 ; stop 1H CPD. 5.5u (p7 ph4):f1 (p14:sp3 ph10):f2 ;========== 4th gradient pulse (homo-spoil). 5u p28:gp4 95u ;========== 200u ;---------- t3(15N) evolution, constant time. (p21 ph21):f3 (p7 ph1):f1 5.5u 4u cpds1:f1 ; start 1H CPD. d30 (p15:sp5 ph10):f2 ; sel 180 pulse for 13CA (off-resonance). d7 9.5u d30 (p24:sp2 ph10):f2 (d12 p21*2.0 ph22):f3 9.5u d7 5.4m d10 (p15:sp5 ph10):f2 ; sel 180 pulse for 13CA (off-resonance). d10 4u do:f1 ; stop 1H CPD. 5.5u (p7 ph4):f1 5.4m ;---------- INEPT 15N-1HN. (p21 ph20):f3 ;========== 5th gradient pulse (homo-spoil). 5u p29:gp5 95u ;========== 200u pl5:f1 ; 1H power -> pl5 (for water flip-back). ;---------- (p5 ph7:r):f1 4u 20u pl1:f1 ; 1H power -> pl1. (p1 ph6):f1 ;========== 6th gradient pulse (echo). 5u p30:gp6 95u ;========== d6 pl5:f1 ; 1H power -> pl5 (for water flip-back). (p5 ph8:r):f1 4u 20u pl1:f1 ; 1H power -> pl1:f1 (d13 p1*2.0 ph3):f1 (p21*2.0 ph20):f3 4u 20u pl5:f1 ; 1H power -> pl5 (for water flip-back). (p5 ph9:r):f1 ;========== 7th gradient pulse (echo). 5u p30:gp6 d6 pl13:f3 ; 15N power -> pl13. 95u setnmr2^0 setnmr0^34^32^33 ;========== ;---------- Acquisition. go=1 ph31 cpd3:f3 ;---------- Loop nest for States or States-TPPI. 50m do:f3 wr #0 if #0 zd 10m ip0 10m dp5 lo to 2 times 2 10m id0 10m ip0*2 10m dp5*2 lo to 3 times l3 15m rd0 15m dp21 lo to 4 times 2 10m id10 10m dd30 10m ip31*2 lo to 5 times l4 ;---------- 30m setnmr2^0 setnmr0^34^32^33 ; gradient-blanking on exit ;phase cycle. ;for 1H ph0=0 ph1=1 ph2=1 3 ph3=2 ph4=3 ph5=2 ph6=0 ph7=(360) 180 ;-x ph8=(360) 0 ;+x ph9=(360) 0 ;+x ;for 13CA/B ph10=0 ph11=0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 ph12=0 0 2 2 ph13=1 3 ph14=0 0 0 0 2 2 2 2 ;for 13C-TOCSY FLOPSY-16 ph15=(360)0 ph16=(360)45 ph17=(360)68 ph18=(360)315 ph19=(360)23 ph25=(360)180 ph26=(360)225 ph27=(360)248 ph28=(360)135 ph29=(360)203 ;for 15N ph20=0 ph21=0 0 0 0 2 2 2 2 ph22=0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 ;for receiver ph31=0 2 2 0 2 0 0 2 2 0 0 2 0 2 2 0 ;cnst21: CO chemical shift (offset, in ppm) ;cnst22: CA chemical shift (offset, in ppm) ;cnst23: CAB chemical shift (offset, in ppm) ;o2p: CAB chemical shift (cnst23) ;gpz1 : 20% p25 ;gpz2 : 40% p19 ;gpz3 : 40% p27 ;gpz4 : 35% p28 ;gpz5 : -45% p29 ;gpz6 : 25% p30