; 15N-T1rho relaxation experiment with sensitivity enhanced (Rance-Kay) HSQC read-out ; for 15N, 2H15N, 15N13C and 2H15N13C labelled proteins ; builts on a sequence by Lakomek et al., JBNMR 2012 ; new version by Tobias Stief October 2022 ;last update Dec 2023 #include #include #include ;#define LABEL_CN ; switch on for 13C labelled samples define list vplist = <$VPLIST> #define TEMP_COMPENSATION "acqt0=0" "in0=inf1*0.5" "d0=10u" "d11=30m" "d26=p7-p1" "DELTA=2.65m" "DELTA1=2.65m-200u-p21" "DELTA2=2.65m-200u-p22" "DELTA3=2.65m-200u-p23" "DELTA4=p25-p19*0.63" "DELTA5=2.65m-p26-200u" "DELTA8=2.65m-200u-p24" "l1=1" "l2=1" # ifdef LABEL_CN "DELTA6=d0*2+26u+p4*4" ;+10u if gp10 active # else "DELTA6=d0*2+p1*2" ;+10u if gp10 active # endif "cnst21=176" "cnst22=56" "cnst18=-800" "spoff4=bf2*((cnst22-cnst21)/1000000)" 1 ze 1m 2 2m do:f3 1m do:f2 1m d11 3 3m 4 1m 5 1m do:f3 1m do:f2 1m BLKGRAD 10u pl1:f1 10u pl4:f2 ;---------temperature compensation------------ # ifdef TEMP_COMPENSATION "p17=p18+1m-vplist" "d17=d1-p17" # endif ;-------delays for spin lock relaxation period------- "d8=p8+vplist*0.25-p15*0.5+1u" "d9=vplist*0.5-p15+2u" "d29=p8+vplist*0.5+2u-p4*2-8u" ;-------- Temperature compensation and d1 recovery delay---------------- #ifdef TEMP_COMPENSATION 10u fq=cnst18(bf ppm):f3 10u pl8:f3 (p17 ph0):f3 ; 15N pulse is applied far off-resonance 10u 10u fq=0:f3 d17 #else d1 #endif 10u fq=cnst21(bf ppm):f2 1m UNBLKGRAD 10u pl7:f3 ;------- kill steady state 15N ------------ (p7 ph0):f3 5u p27:gp7 200u ;------- first INEPT Hz-> 2HxNz ------------------------- (p1 ph0):f1 5u p21:gp1 200u DELTA1 (center(p1*2 ph0):f1 (p7*2 ph0):f3) DELTA1 5u p21:gp1 200u ;------- rephase 2HxNz to Nz----------------------------- (p1 ph5):f1 (p7 ph0):f3 5u p22:gp2 200u DELTA2 (center (p1*2 ph0):f1 (p7*2 ph0):f3) DELTA2 5u p22:gp2 200u (p7 ph6):f3 ; phase-cycle Nz, -Nz for Freeman-Hill decay 5u ;-------------------------------------------------------- (p1 ph2):f1 ; purge pulse to kill any residual HzNz 5u p28:gp8 100u 50u pl8:f3 ; change to pl0:120dB for Avance I or DRX systems 50u pl0:f1 ;------ N15 T1rho relaxation period with shaped 180 H pulses for CCR compensation ----------- # ifdef LABEL_CN (d8 p15:sp5 ph0 d9 p15:sp5 ph0 d8):f1 (p8:sp8 ph0 4u pl8 vplist ph0 p8:sp9 ph0):f3 (d29 p4*2 ph0 3u 10u fq=cnst22(bf ppm) 3u p4*2 ph0 10u fq=cnst21(bf ppm)):f2 #else (d8 p15:sp5 ph0 d9 p15:sp5 ph0 d8):f1 (p8:sp8 ph0 4u pl8 vplist ph0 p8:sp9 ph0):f3 #endif ;------------Cleaning Gradient------------------------ 5u p29:gp9 100u 100u ;fq=0:f1 ;---------- Echo- Antiecho encoding------------------------ 77 3u pl7:f3 3u pl4:f2 3u pl1:f1 (p7 ph7):f3 DELTA6 ;compensation for d0 15N evolution DELTA5 190u p26:gp6*EA 10u (center (p1*2 ph0):f1 (p7*2 ph7):f3) 5u p26:gp6*EA*-1 195u DELTA5 ; t1 evolution --------------------------------------------- d0*0.5 ;gron10 5u groff d0*0.5 ;gron10*-1 5u groff #ifdef LABEL_CN (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 ;--------first INEPT for sensitivity-enhanced back transfer---------------------------- (center (p1 ph0):f1 (p7 ph8):f3) 5u p23:gp3 200u DELTA3 (center(p1*2 ph0):f1 (p7*2 ph0):f3) DELTA3 5u p23:gp3 200u ; ---second INEPT ----------------------------------------- (center (p1 ph1):f1 (p7 ph1):f3) 5u p24:gp4 200u DELTA8 (center(p1*2 ph0):f1 (p7*2 ph0):f3) DELTA8 5u p24:gp4 200u (d26*0.5 p19 ph0:r):f1 ; ------------------------spin-echo with decoding gradient EA 998 245u DELTA4 (p1*2 ph0):f1 5u p25:gp5 200u 999 5u 5u pl30:f2 10u pl31:f3 20u BLKGRAD go=2 ph31 cpds3:f3 ;cpd2:f2 200u 400u do:f3 400u do:f2 d11 wr #0 if #0 zd 1m 2m vplist.inc lo to 3 times l6 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 ph2=2 ph5=1 ph6=1 1 3 3 ph7= 1 3 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) ; 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: 1000 dB ;p15: 1000u (@ 1200 MHz) 180 deg IBurp2 pulse on 1H (sp15) ;sp5: 180 deg IBurp2 pulse on 1H (sp15) ;spnam5: IBurp2 ;spoffs5: 4680Hz @ 1200 MHz (8.6 ppm) , should be centered in amide region but not touch the water ; 13C pulses ;p4: 13CO selective 180 deg (11.850*2us @ 1200 MHz) @pl4 ;pl4: 13C 90 deg ;sp4: 13CA selective 180 deg (11.850*2us @ 1200 MHz) ;15N pulses ;p7 : 90 deg hard 15N pulse @pl7 ;pl7: 15N 90 deg ;pl8: 15N spin-lock power ;pl0 1000 dB ;sp8 @pl8 spin-lock power ;sp9 @pl8 spin-lock power ;spnam8 AHP TanhTan (1st half) ;spnam9 AHP TanhTan (2nd half) ;spoal8 1 ;spoal9 0 ;CPDPRG3: garp (15N decoupling) ;pcpd3: 15N decoupling (200u @pl31) ;pl31: 15N decoupling power ; gradients ;p21: 200u ;p22: 200u ;p23: 800u ;p24: 200u ;p25: 201u Echo/Anti-echo decoding gradient ;p26: 1000u Echo/Anti-echo half-encoding gradient ;p27: 200u ;p28: 200u ;p29: 200u ;for z-only gradients ;gpz1: 1% ;gpz2: 2% ;gpz3: 31% ;gpz4: 11% ;gpz5: 50% ;gpz6: 50% ;gpz7: 50% ;gpz8: 5% ;gpz9: 10 % optional ;gpz10: 0.5% optional prevent t1 radiation damping ;gpnam1 SINE.20 ;gpnam2 SINE.20 ;gpnam3 SINE.20 ;gpnam4 SINE.20 ;gpnam5 SINE.20 ;gpnam6 SINE.20 ;gpnam7 SINE.50 ;gpnam8 SINE.20 ;gpnam9 SINE.20