;HCAN, gradient enhanced, deuterium decoupled ; ;Bruker DMX Avance, XwinNMR version ;separate include file bits.mo ;separate gradient program hcacon-ge.r ;for measurement of two-bond amide deuterium effects on carbon-alpha ;for use with 15N-13C-labeled proteins in 50% D20/50% H2O ;States-TPPI, gradient enhancement ;Ref: Ottiger, M. & Bax, A. (1997) J. Am. Chem. Soc. 119, 8070-8075. ;implemented by Marcel Ottiger ;ottiger@nih.gov ;last modified 6/3/97 ;BMRB Pulse Sequence Accession Number: 14 #include "bits.mo" #define NITROGEN ; uncomment for 2D without 15N evolution #define CARBON ; uncomment for 2D without 13C evolution #define NITROGEN_LOOP l1 #define CARBON_LOOP l2 ;#define PULSE_CHECK ; set p17 to 2*p7 => zero signal ;******************* Pulse definitions ************************ ;Proton (H = hydrogen frequency on water) ;p1 = proton 90 at pl1 ;p30= proton 90 at pl30 (cpds1: waltz16.30 decoupling) ;CA (C1 = carbon frequency 57.5 ppm) ;p3 = sel. carbon 90, null for C': 54us at pl3 [=sqrt(15)/(4*Omega)] ;p4 = 1/2 carbon 180, null for C': 24.2us at pl4 [=sqrt(3)/(4*Omega)] ;p31= carbon 90 at pl31 (cpd2: waltz16.31 decoupling) ;C' (C2 = carbon frequency 176 ppm) ;p5 = shaped sinc1.0 90 pulse, ~100us at sp1 ;p6 = shaped sinc1.0 180 pulse (= p5*2) ;Nitrogen (N= nitrogen frequency 118ppm) ;p7 = nitrogen 90, at pl7 (** MUST ** be shorter than p3!) ;p17 for pulse check ;p29= nitrogen 90 at pl29 (cpds2: waltz16.29 decoupling) ;Deuterium decoupling CW at pl8 ;Gradients #define GRAD20(ampl) p20:ngrad:c34 ; 5.0m x,y #define GRAD21(ampl) p21:ngrad:c34 ; 1.5m x #define GRAD22(ampl) p22:ngrad:c34 ; 0.7m y ;#define GRAD23(ampl) p23:ngrad:c34 ; 0.5m x not used #define GRAD24(ampl) p24:ngrad:c34 ; 1.56m z optimize for max signal #define GRAD25(ampl) p25:ngrad:c34 ; 0.5m x #define GRAD26(ampl) p26:ngrad:c34 ; 0.5m y #define GRAD27(ampl) p27:ngrad:c34 ; 0.25m z #define GRAD28(ampl) p28:ngrad:c34 ; 0.15m -z ;"cnst20= 50" ;"cnst21=-50" ;******************* Evolution Incrementation ********************** ;Nitrogen incrementation: in10 = 1/2sw ;Carbon incrementation: in20 = in21 = 1/4sw (!!!!) ;******************* Delay/Pulse calculations ************************ "d3=p3/2 - p1/2" "d4=p4 - p1" "d5=8m" ; universal dd, id, ip delay "d7=p7-p4" "d10=in10/2 -p4 -p5 -p7*0.6366 -15u" ;15N-evolution (initial t1/2) "d15=50m" "d20=3.74m -p5" ; 13C-evolution (t2/4 +const) "d21=7.138m -p5" ; 13C-evolution (initial T2/4) "d24=3.2m - p24" "d25=1.5m - p25" "d26=1.5m - p26" "d27=0.5m - p27" "d28=0.5m - p28" "p6=p5*2" #ifndef PULSE_CHECK "p17=p7" #endif ;*********************** Pulse program ************************** 100u RESET 1 ze 2 d15 d5*2 3 d5*2 4 d5*3 5 d5*3 ;__________________________________________________________________Preparation 6 100u RESET 5u pl1:H 5u pl7:N 1m LOCK_ON d1 ; relaxation delay 1m LOCK_OFF (p7 ph0):N ; get rid of Boltzmann 2u GRAD20(cnst20) 5m ;_______________________________________________________First INEPT to C-alpha (p1 ph0):H 1.5m pl4:C1 (d4 p1*2 ph0):H (p4*2 ph0):C1 1.5m (p1 ph1):H 2u GRAD21(cnst20) 1.5m pl3:C1 ;________________________________________________________________ INEPT to 15N (p3 ph0):C1 3.4m pl30:H 9.88m cpds1:H ; Proton decoupling on 1m pl4:C1 (d7 p4*2 ph0):C1 (p7*2 ph0):N ; center pulses 14.28m pl3:C1 (p3 ph2):C1 2u GRAD22(cnst20) 0.7m pl8:D 5u DEUT_ON ; deuterium decoupler on 2u cw:D ;________________________________________________________________15N-Evolution (p17 ph10):N ; p17 for pulse check d10 pl4:C1 (p4*2 ph0):C1 30u (p6:sp1 ph0):C2 d10 pl3:C1 (p3 ph0):C1 (p7 ph0):N ; pulses start together (p3 > p7) ;_____________________________________________________________13C-CT-Evolution 2u do:D d21 DEUT_OFF (p6:sp1 ph0):C2 d21 pl4:C1 2u (d7 p4*2 ph3):C1 (p7*2 ph0):N ; center pulses 3.4m d20 (p6:sp1 ph0):C2 d20 pl3:C1 0.2m do:H ; proton decoupling off GRAD24(cnst20) d24 pl1:H (d3 p1 ph0):H (p3 ph20):C1 ;____________________________________________________________________Rance-Kay 2u GRAD25(cnst20) d25 pl4:C1 (d4 p1*2 ph0):H (p4*2 ph0):C1 2u GRAD25(cnst20) d25 pl3:C1 (d3 p1 ph1):H (p3 ph1):C1 2u GRAD26(cnst20) d26 pl4:C1 (d4 p1*2 ph0):H (p4*2 ph0):C1 2u GRAD26(cnst20) d26 (d3 p1 ph0):H 2u GRAD27(cnst20) d27 (p1*2 ph0):H 2u GRAD28(cnst21) d28 pl31:C1 go=2 ph31 cpd2:C1 ; acquisition, C-alpha decoupled d15 do:C1 wr #0 if #0 zd ;_____________________________________________________________________________ #ifdef CARBON d5 ip20 d5 ip20 lo to 3 times 2 d5 id20 d5 dd21 lo to 4 times CARBON_LOOP #endif #ifdef NITROGEN d5 rd20 d5 rd21 d5 ip10 lo to 5 times 2 d5 id10 d5 ip31 d5 ip31 lo to 6 times NITROGEN_LOOP #endif 1u do:C1 1u do:H 100u RESET 1m LOCK_ON exit ph0=0 ph1=1 ph2=0 0 0 0 2 2 2 2 ph3=0 1 2 3 ph10=0 0 2 2 ; phase for quad det 15N ph20=0 ; phase for Rance-Kay 13C ph31=0 2 2 0 2 0 0 2