;hncacbse.ref ;ver 06-03-02 ;3D HNCACB, gradient enhanced with Watergate reverse INEPT (3-9-19), ;minimal H2O saturation and optional 2H decoupling ;Yamazaki et al., J. Am. Chem. Soc. 1994, 116, 11655-11666 ;Wittekind and Mueller, J. Magn. Reson. B 101 201-205 (1993) ;Mori et al., J. Magn. Reson. B 108, 94-98 (1995) ;Bruker Avance/Xwin-nmr version ;Written up by F. Abildgaard, NMRFAM (abild@nmrfam.wisc.edu) ;UTHSCSA setup by A. Hinck, 1/02 ;NOTE1 - Follow the guidelines below for setting up this experiment ;Unless you fully understand what you are doing, do NOT proceed beyond ;the point indicated ;NOTE2 - In the header section that follows, anything between double quotes ;is something you can type at the XWINNMR command line ;NOTE3 - Anything followed by a "s" means a time in seconds, anything followed ;by a "m" means a time in milliseconds, and anything followed by a "u" ;means a time in microseconds ; ;Set the options below to tailor the pulse program for your needs ;#define ONE_D ; uncomment for 1D experiment #define N15_EVOL ; comment out for 2D w/o N15 evolution ;#define C13_EVOL ; comment out for 2D w/o C13 evolution #define SHAPED ; uncomment if shaped flip-back pulse ;#define H2_DEC ; uncomment to enable H2 decoupling ;#define SIDECHN ; uncomment for delays optimized for side-chain amide corr #define EXPTCORR ; uncomment if you want "expt" to report ; ; the correct expt time (works with XWIN-NMR 2.x and above) ; ;Set the channel assignments to match your hardware settings in "edasp" #define H f1 #define C f2 #define N f3 #define D f4 ; ;Gradient settings ;Note1 - grad times are preset, you do not need to adjust these ;Note2 - set grad params according to table below, note integer in first ;column is substituted to yield the parameter that is to be adjusted ;example for grad# 0, set "gpnam0 sine.64", "gpx0 0", "gpy0 0", "gpz0 6" ; ;grad# gpnam# gpx# gpy# gpz# ;1 sine.64 0 0 6 ;2 sine.100 0 0 15 ;3 sine.100 35 0 20 ;4 sine.100 0 0 -8 ;5 sine.100 0 0 15 ;6 sine.100 0 0 15 ;7 sine.64 0 0 12 ;8 sine.10 0 0 29 ;9 sine.10 0 0 44 ;1H pulses, set carrier to H2O ;p1 - square 90 degree 1H @ pl1, typically p1 = 8u @pl1 = -2dB ;p10 - shaped 90 degree 1H @ sp1 (if SHAPED defined - see above) ; typically p10 = 2m @ sp1 = 46.4 dB, "spnam1 seduce1.jc", "spoffs1 0" ;p10 - square 90 degree 1H @ pl10 (if SHAPED not defined - see above) ; typically p10 = 2m @ pl10 = 47 dB ;p11 - square 90 degree 1H @ pl11, used for 1H cpd, ; typically, p11 = 40u @ pl11 = 13.8 dB ; "cpdprg1 waltz16" or "cpdprg1 dipsi2" ;pcpd1 - set this to be equal to p11 ;15N pulses, set carrier to 118ppm ;p2 - square 90 degree 15N @ pl2, typically p2 = 40u @ pl2 = -5dB ;pcpd2 - square 90 degree 15N @ pl12, used for 15N cpd ; typically pcpd2 = 175u @ pl12 = 7.9dB ; "cpdprg2 waltz16" ; ;13C pulses, set carrier to 43 ppm ;p3 - square 90 degree 13C @ pl3, typically p3 = 14u @ pl3 = -3dB ;pcpd3 - shaped 90 degree @ sp3, used for decoupling of CO ; typically pcpd3 = 299u @ sp3 = +10.9 dB (for seduce1c6.jc, see below) ; "spoffs3 0", "spnam3 seduce1c5.jc" or "spnam3 seduce1c6.jc" ; "cpdprg3 waltz16sp3" ; Note, the seduce1c5.jc and seduce1c6.jc shaped pulses give no ; excitation on resonance; Instead their excitation maximum falls ; at either +/- 5/pw90 (for seduce1c5.jc) or +/- 6/pw90 (for seduce1c6.jc) ; Hz away from the carrier ; where pw90 is the 90 degree shaped pulse time (this is equivalent to pcpd3 ; in this particular case). Since the 13C carrier is at 43 ppm and we want to ; decouple at the carbonyls (these are at ~ 176 +/- 3 ppm), the offset in this ; case will be: (176 - 43 ppm)*150.89 Hz/ppm = 20068 Hz. Thus, if you ; choose to use seduce1c6.jc, you would want to set the 90 degree pulse (or ; pcpd3 in this case) to 5/20068Hz = 299u ; Owing to the fact that this pulse gives maximal excitation off-resonace, ; when calibrating it is necessary to shift the 13C carrier by the calculated ; offset, to set the pulse time accordingly, and to then adjust the shaped ; power level to give a null. In the example above, the 13C carrier would ; be shifted by -20068 Hz, the pulse time would be set to 299u, and the power ; level would be adjusted (nominally) to 10.9 dB ;2H settings (OPTIONAL, H2_DEC option above), set carrier to 4.5ppm ;p25 - square 90 degree 2H @ pl15, typically xxu @ pl15 = xxdB, used for 2H cpd ; "cpdprg5 waltz16" ;pcpd5 - set equal to p25 ;15N Shift Evolution ;in10 - in10=1/(2*SW), typically in10 = 300u ;in30 - in30=1/(2*SW), typically in30 = 300u ;l4 - number of complex pts, max is (d10/in10)+1, typically l4 = 39 ;13C Shift Evolution ;in0 - in0=1/(2*SW), typically in0=49.5u ;l6 - number of complex pts, typicaly l6=75 ;cnst0 - set to 0 preferably or 1 to make d0 the smallest possible ; you can judge how to set cnst0 by looking ; at the expression for d0 below (no touching!); the only requirment is ; that d0 should be the smallest possible positive value (negative ; values are (obviously) not allowed). ; typically, cnst0 = 1 ;Other settings ;d1 - recycle time, typically d1 = 1s ;d3 - dephasing delay to generate Ca/Cb magnetization, ; typically, d3 =3.5m for Ca/Cb or d3 = 6.8m for Cb only ;pl0 - unused power level, set to +120dB ;d21 - delay used for 3-9-19 water suppression, set to 1/(2*dNu), where ; dNu=Nu(NH) - Nu(H2O), dNu typically 2100 Hz, d21 typically 238u ;ns - set to a multiple of 8 ;ds - set to a multiple of 8 ;1 td - set to l4*l6*4 (if both C13_EVOL and N15_EVOL defined) ;1 td - set to l4*4 (if N15_EVOL defined and C13_EVOL undefined) ;1 td - set to l6*4 (if C13_EVOL defined and N15_EVOL undefined) ;OPTIONAL Settings (adjustments may not be necessary) ;phcor21 - adjust as necessary for phase difference between p10@sp1 (if ;SHAPED defined) or p10@pl10 (if SHAPED not defined), typically phcor21 = 0 ;15N Dimension Processing ;Conversion: Dim = y, zMODE = Complex, aq2D = States ;Processing: ;|nmrPipe -fn SP -off 0.45 -end 0.98 -pow 1 -c 0.5 \ ;|nmrPipe -fn ZF -size 128 \ ;|nmrPipe -fn FT -neg \ ;|nmrPipe -fn PS -p0 0.0 -p1 0.0 -di -verb \ ;|nmrPipe -fn POLY -auto -ord 1 \ ;13C Dimension Processing ;Conversion: Dim = z, yMODE = Complex, aq2D = States ;Processing (depends on the value of cnst0; shown below is example for cnst0 = 1) ;|nmrPipe -fn ZF -pad 1 \ ;|nmrPipe -fn RS -rs 1 -sw \ ;|nmrPipe -fn LP -before -pred 1 \ ;|nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 1.0 \ ;|nmrPipe -fn ZF -size 512 \ ;|nmrPipe -fn FT \ ;|nmrPipe -fn PS -p0 -90.0 -p1 180.0 -di -verb \ ;Processing (depends on the value of cnst0; shown below is example for cnst0 = 0) ;|nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 1.0 \ ;|nmrPipe -fn ZF -size 512 \ ;|nmrPipe -fn FT \ ;|nmrPipe -fn PS -p0 -90.0 -p1 180.0 -di -verb \ ;Testing ;C13 plane gives the strongest signal since N15 undergoes 0/0 phase shift ;for the first N15 point ;N15 plane gives a weak signal since Ca/Cb signals are of opposite sign and ;tend to cancel. Additionally, C13 undergoes either -90/180 or -270/540 ;phase shift for cnst0=0 and cnst0=1, respectively, which causes significant ;dephasing for the first fid ;PRESET DELAYS - DO NOT ADJUST ANYTHING BEYOND THIS POINT ; "p15=500u" "p16=1.0m" "p17=700u" "p18=1.0m" "p19=600u" "p20=100u" ; ;sanity checks #ifdef ONE_D #undef N15_EVOL #undef C13_EVOL #endif ; aqseq 321 ; define delay TAUA1 define delay TAUA2 define delay TAUB define delay TAUC define delay TAUC1 define delay TAUF1 define delay TAUF2 define delay TAUW define delay TN define delay C_H_N define pulse H1_90 define pulse H1_180 define pulse H1_S90 define pulse N15_90 define pulse N15_180 define pulse CAB_90 define pulse CAB_180 "d11=100m" ;disk i/o "d12=10u" ;power switching etc. "d13=5u" ;a short delay "d28=150u" "d14=60u" ;ip,id etc. "d16=300u" ;gradient recovery "d17=50u" ;short gradient recovery "H1_90=p1" "H1_180=p1*2" "H1_S90=p10" "N15_90=p2" "N15_180=p2*2" "CAB_90=p3" "CAB_180=p3*2" "TAUW=d21" "TAUA1=2.25m-p15-d16-d13" "TAUA2=2.25m-TAUW*2.5-H1_90*2.385-p17-d16-d13" #ifdef SIDECHN "TAUB=2.7m" "TN=20.0m" #else "TAUB=5.4m" "TN=12.4m" #endif "TAUC=TN-CAB_180" "TAUC1=TAUC+CAB_180-TAUB-p11-d13-d12" #ifdef H2_DEC "TAUF1=d3-p20-d17-p11-p25-d12*3-d13*7" "TAUF2=TAUF1-d12-d13" #else "TAUF1=d3-p20-d17-p11-d12*3-d13*7" "TAUF2=TAUF1-d13" #endif "d10=TN-d13*2-d12" "d30=TN-TAUB-CAB_180-p11-d12*2-d13*5" #ifdef C13_EVOL "d0=((cnst0*2+1)*in0-CAB_90*1.273-N15_180-d12*2-d13*4)/2" #endif "d22=(TAUW/2)-N15_90" "C_H_N=N15_90-H1_90" #ifdef EXPTCORR "d31=2*TAUA1+2*TAUB+TAUC1+TAUC+2*TAUF1+2*TAUF2+2*TAUA2+4*TAUW" #endif #define H1_DEC_ON d13 \n d12 pl11:H \n p11:H ph1 \n d13 cpds1:H #define H1_DEC_OFF d13 do:H \n p11:H ph3 \n d13 \n d12 pl1:H #define H1_DEC_OFF_Y d13 do:H \n p11:H ph1 \n d13 \n d12 pl1:H #ifdef H2_DEC #define H2_DEC_ON d12 pl15:D \n p25:D ph1 \n d13 cpds5:D #define H2_DEC_OFF d13 do:D \n p25:D ph3 #else #define H2_DEC_ON d13 #define H2_DEC_OFF d13 #endif #define SED_ON d13 \n d12 pl0:C \n d13 cpds3:C #define SED_OFF d13 do:C \n d12 pl3:C \n d13 #include #include 1 ze 2 d11 do:N d14 3 d14 d14 d14 d14 4 d14 d14 d14 5 d14 d14 d14 d14 6 d14 H2_LOCK d14 LOCKH_OFF #ifdef EXPTCORR #include #endif d1 pl1:H pl2:N d14 LOCKH_ON d14 H2_PULSE (N15_90 ph0):N d13 p17:gp6 ;400u, 10G/cm d16 (H1_90 ph0):H d13 p15:gp1 ;500u, 4G/cm d16 TAUA1 (C_H_N H1_180 ph0):H (N15_180 ph0):N TAUA1 d13 p15:gp1 ;500u, 4G/cm d16 (H1_90 ph1):H d13 #ifdef SHAPED d12 pl0:H (H1_S90:sp1 ph21):H ;2ms SEDUCE-1 90 H1 pulse at phase x #else d12 pl10:H (H1_S90 ph21):H ;2ms 90 H1 pulse at phase x #endif d13 p16:gp2 ;1.0m, 10G/cm d16 SED_ON (N15_90 ph11):N TAUB H1_DEC_ON TAUC1 SED_OFF (N15_180 ph0):N d13 (CAB_180 ph0):C SED_ON TAUC (N15_90 ph0):N H1_DEC_OFF SED_OFF d13 p18:gp4 ;1.0m, -5G/cm d16 H2_DEC_ON H1_DEC_ON (CAB_90 ph22):C SED_ON TAUF1 H2_DEC_OFF H1_DEC_OFF SED_OFF p20:gp8 ;100u, 20G/cm d17 (CAB_180 ph0):C d13 p20:gp8 ;100u, 20G/cm d17 SED_ON H1_DEC_ON H2_DEC_ON TAUF2 SED_OFF (CAB_90 ph1):C #ifdef C13_EVOL SED_ON d0 (N15_180 ph0):N d0 SED_OFF #else d28 ;d13 #endif (CAB_90 ph23):C SED_ON TAUF1 H2_DEC_OFF H1_DEC_OFF SED_OFF p20:gp9 ;100u, 30G/cm d17 (CAB_180 ph0):C d13 p20:gp9 ;100u, 30G/cm d17 SED_ON H1_DEC_ON H2_DEC_ON TAUF2 SED_OFF (CAB_90 ph24):C H2_DEC_OFF H1_DEC_OFF p19:gp5 ;600u, 10G/cm d16 SED_ON H1_DEC_ON (N15_90 ph13):N d10 SED_OFF (N15_180 ph14):N d13 (CAB_180 ph0):C SED_ON d30 H1_DEC_OFF TAUB (N15_90 ph0):N SED_OFF d13 p15:gp7 ;500u, 8G/cm #ifdef SHAPED d16 pl0:H (H1_S90:sp1 ph21):H ;2ms SEDUCE-1 90 H1 pulse at phase x #else d16 pl10:H (H1_S90 ph21):H ;2ms 90 H1 pulse at phase x #endif d13 d12 pl1:H (H1_90 ph2):H d13 TAUA2 pl1:H p17:gp3 ;700u, 40G/cm d16 (H1_90*0.231 ph1):H TAUW (H1_90*0.692 ph1):H TAUW (H1_90*1.462 ph1):H d22 (N15_180 ph0):N d22 (H1_90*1.462 ph3):H TAUW (H1_90*0.692 ph3):H TAUW (H1_90*0.231 ph3):H d13 p17:gp3 ;700u, 40G/cm d16 pl12:N TAUA2 go=2 ph31 cpds2:N #ifdef ONE_D d11 do:N wr #0 #else d11 do:N wr #0 if #0 zd #endif #ifdef N15_EVOL d14 ip13 lo to 3 times 2 d14 dd10 d14 id30 d14 ip31 d14 ip31 lo to 4 times l4 d14 rd10 d14 rd30 #else d14*7 #endif #ifdef C13_EVOL d14 ip23 d14 ip24 lo to 5 times 2 d14 id0 d14 ip31 d14 ip31 lo to 6 times l6 #endif d14 H2_LOCK d14 LOCKH_OFF exit ph0=0 ph1=1 ph2=2 ph3=3 ph11=0 2 ph13=0 ph14=0 0 0 0 2 2 2 2 ph22=0 0 2 2 ph23=1 1 1 1 3 3 3 3 ph24=0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 ph21=(360)0 ; phase x, adjust for any phase diff. between pl1 and sp1/pl110 ph31=0 2 2 0 0 2 2 0 2 0 0 2 2 0 0 2