; 15N1H HetNoe relaxation experiment with sensitivity enhanced (Rance-Kay) HSQC read-out ; for 15N, 2H15N, 15N13C and 2H15N13C labelled proteins ; written by NL 10/27/11 ;last update by TS 04/24 ; see footnotes #include #include #include #define LABEL_CN ; switch on for 13C labelled samples "in0=inf1*0.5" "acqt0 = 0" "d0=10u" "d11=30m" "d26=p7-p1" "DELTA2=2.65m" "DELTA3=2.65m" "DELTA4=p24-p19*0.63" "DELTA5=2.65m-p25-200u" "DELTA7=2.65m" "cnst22=176" "cnst21=56" "l1=1" "l2=1" # ifdef LABEL_CN "DELTA6=35u+p4*4" "d25=20m-p15*0.5-p4*4-5u" # else "DELTA6=30u" "d25=20m-p15*0.5-p4*4-5u" # endif "cnst21=176" "cnst22=56" "cnst23=8.5" "spoff4=bf2*((cnst22-cnst21)/1000000)" "spoff18=bf3*((cnst18)/1000000)" "d8=p1" 1 ze 1m 2 3m do:f3 d11 1m LOCKH_OFF 3 3m 4 1m 5 1m do:f3 1m BLKGRAD 10u pl1:f1 10u pl4:f2 10u pl7:f3 (p7 ph0):f3 ; 90N pulse before d1 d1 ;--------------- 1H saturation period----------------------- 10u fq=cnst23(bf ppm):f1 if "l2 == 1" goto 9 8 11m (p1*2 ph0):f1 11m lo to 8 times l8 goto 10 9 11m d8*2 11m lo to 9 times l8 10 10u fq=0:f1 10u UNBLKGRAD ;---------- Echo- Antiecho encoding------------------------ (p7 ph7):f3 ;goto 999 DELTA6;compensation for d0 15N evolution DELTA5 190u p25:gp5*EA 10u (center (p1*2 ph0):f1 (p7*2 ph7):f3) 10u p25:gp5*EA*-1 190u DELTA5 ; t1 evolution --------------------------------------------- 89 d0*0.5 gron1 5u groff d0*0.5 gron0 5u groff # ifdef LABEL_CN ;(center (p1*2 ph0):f1 (p4*2 ph0 3u 3u pl2 p4*2:sp4 ph0):f2) (center (p1*2 ph0):f1 (p4*2 ph0 3u 10u fq=cnst22(bf ppm) 3u p4*2 ph0 10u fq=cnst21(bf ppm)):f2 ) # else (p1*2 ph0):f1 # endif d0 ;goto 999 ;--------Rance-Kay transfer back ---------------------------- 98 (center (p1 ph0):f1 (p7 ph8):f3) DELTA2 gron2 10u groff (center(p1*2 ph0):f1 (p7*2 ph0):f3) DELTA2 gron2 10u groff ; ---second INEPT ---------------------------------------- (center (p1 ph1):f1 (p7 ph1):f3) ;DOUBLE 90 DELTA3 gron3 10u groff (center(p1*2 ph0):f1 (p7*2 ph0):f3) DELTA3 gron3 10u groff (d26*0.5 p19 ph0:r):f1 998 245u DELTA4 (p1*2 ph0):f1 5u p24:gp4 200u 999 5u 10u pl31:f3 20u BLKGRAMP go=2 ph31 cpds3:f3 1m do:f3 d11 wr #0 if #0 zd 1m LOCKH_OFF 2m iu2 lo to 3 times 2 1m ip8*2 1m igrad EA 1m ru2 lo to 4 times 2 1m id0 lo to 5 times l3 1m do:f3 1m BLKGRAD exit ph0=0 ph1=1 ;check right phase for Boltzmann !!!!! ph2=2 ph7= 1 3 3 1 ph8= 0 ph31=1 3 3 1 ;-------------NOTES---------------------- ;o1p = 4.7 ppm ;o2p=176 ppm (CO) ;o3p=119 ppm ;NS=4*n ;in0=inf/2 ;SW=1/(2*in0) ;echo-antiecho in N15 (process as Complex in NmrDraw before splitting the spectra) ;l3: # complex points in indirect dimension ;l8: loopcounter for 1H saturation; duration of saturation = l8 * 22m ; 1H pulses ;p1: 90 deg hard 1H pulse @pl1 ;p19 last 90 deg hard 1H pulse @pl1, can be adjusted for improving water-supression ;pl1: 1H 90 deg ;pl0: 120 dB ; 13C pulses ;p4: 90 deg pulse length (11.850 us @ 1200 MHz) for selective 13CO 180 deg @pl4 ;pl4: 13C 90 deg ;pl30: cpd decoupling of f2 if desired, default 1000 db ;15N pulses ;p7 : 90 deg hard 15N pulse @pl7 ;pl7 :15N 90 deg ;CPDPRG3: garp (aq 15N decoupling) ;pcpd3: 15N decoupling (200u @pl31) ;pl31: 15N decoupling power ; gradients ;p20: 1000u ;p21: 200u ;p24: 201u Echo/Anti-echo decoding gradient ;p25: 1000u Echo/Anti-echo half-encoding gradient ;for z-only gradients ;gpz0: 1.5% ;gpz1: -1.5% ;gpz2: -3% ;gpz3: 0.5% ;gpz4: 40% ;gpz5: 40% ;gpz10: 1% ;gpz11: 2% ;gpz20: 28% ;gpz21: 50% ;gpnam4 SINE.20 ;gpnam5 SINE.20 ;gpnam20 SINE.50 ;gpnam21 SINE.20