; M.CDCAncoCAHA ; ; cdcacaha_pro ; sequential assignment of prolines and poly-proline stretches in 13C/15N-labelled proteins ; 3D CDCA(NCO)CAHA ; (HDCD/HACA)->N->CO->CA->HA transfer experiment ; EMBL Heidelberg: M.J.Bottomley, M.J.Macias, Z.Liu & M.Sattler ; J. Biomol. NMR (1999) 13, 381-385. ; Two constant time evolutions; first for CD/CA(i), then for CA(i-1) ; Editing for prolines is achieved via selection of N not bound to HN for H2O samples ; CA(i-1) --> HA(i-1) transfer using E/AE and sensitivity enhancement ; CA/CD-selective pulses to refocus 1J(CA,CB)/1J(CD,CG): p12=1.25ms RE-BURP.256 ; channels: ; F1 1H 4.7 ppm ; F2 13CD/CA 56 ppm ; F3 15N 118 ppm ; evolutions: ; F1 13CA (i-1) ; F2 13CA/CD (i) ; F3 1HA (i-1) ; set FnMODE F2 = F1 = undefined ; GRDPROG 4sine0eaxyz_pro (-80 70 +/-90 90.3) ; GRADIENT(cnstx) using p16 ; GRADIENT2(cnstx) using p19=p16/4 #include #include #include #define PI_N p5 ph8 3u p6 ph0 3u p5 ph8 "acqt0=0" "in0=inf1/2" "in20=in0" "in10=inf2/2" "in30=in10" "CEN_HN1=(p6*2+6u-p2)/2" "CEN_CN1=(p14-p6)/2" "CEN_CN2=(p14-p6*2-6u)/2" "CEN_HC1=(p14-p2)/2" "p19=p16/4" "p2=p1*2" "p6=p5*2" "d8=p1+p13-p14" "d9=p13-p14" "d29=p19+350u" "d21=5.4m" ; 1/2J(H,N) "d4=1.8m" ; 1/4J(H,C) "d24=d4" ; 1/4J(H,C) "d19=2.0m" ; ~1/4J(H,C) "d22=7m" ; CA -> N "d23=14m" ; N -> CO "d15=4.5m" ; CO -> CA "d26=d22-p14" "d10=3u+in10/2" ; +in10/2 "d30=d26+3u-in10/2" ; -in30/2 ;;"d10=3u" ;;"d30=d26+3u" "d27=3u+d26+p14+CEN_CN2" "d25=d23-d21" "d7=d23+3u-p6*2-6u-d15" "d0=3u" "d20=3.51m" ; 1/2J(CO,CA) "d18=3.5m-p16" ; 1/2J(H,C) "d5=d20-p16-d18-6u" "l0=td1/2" "l10=td2/2" ;============================================================================== ; set offsets for selective pulses, considering frequency jump by "fq" command "cnst30=o2/bf2" ; = o2p (13C) "cnst32=cnst22-cnst30" ; additional offset after freq jump with cnst22 "cnst33=cnst23-cnst30" ; additional offset after freq jump with cnst23 ;shaped pulses at frequency=cnst22 "spoff4=bf2*((cnst21-cnst32)/1000000)-o2" ;shaped pulses at frequency=cnst23 "spw14=spw4" ;CO selective "spoff14=bf2*((cnst21-cnst33)/1000000)-o2" "spoff5=bf2*((cnst21-cnst33)/1000000)-o2" "spoff6=bf2*((cnst21-cnst33)/1000000)-o2" aqseq 312 1 d11 ze d11 pl13:f3 pl2:f2 2 d1 do:f2 do:f3 6m 3 20m 4 12m 5 20m 6 20u pl1:f1 20u pl3:f3 ; 100u fq2:f2 ; 13C carrier at 56ppm (for CD/CA selection) 30u fq=cnst22(bf ppm):f2 (p1 ph0):f1 ; ----------------- HD->CD, HA->CA d4 (p14:sp3 ph13):f2 (CEN_HC1 p2 ph0):f1 d4 (p1 ph2):f1 3u 100u UNBLKGRAD p16:gp0*EA ; EA for better water suppression d16 pl11:f1 (p13:sp1 ph3):f2 ; ----------------- CA --> N 3u (d19 3u cpds1):f1 (d10 p14:sp4 ph0 d26 p12:sp12 ph9 d30 p14:sp4 ph0):f2 (d10 d27 PI_N):f3 ; In d19 2HzCy --> Cx, then start 1H decoupling ; p12 for CA/CD selective refocusing, p14 for C' decoupling then BSP 3u (p13:sp2 ph0):f2 3u do:f1 ; 1H decoupling off (enables proline selection) (p5 ph4):f3 ; ----------------- N --> CO; d21 ; 1/2J(N,H) d25 cpds1:f1 ; 1H decoupling on (destroys antiphase operators of non-prolines) (p14:sp3 ph0):f2 (p6 ph0):f3 (p14:sp4 ph0):f2 d23 ; ~ 1/4J(N,C') (p5 ph0):f3 3u do:f1 ; 1H decoupling off p16:gp1 d16 fq=cnst23(bf ppm):f2 ; Change 13C carrier to 59 ppm (for CA selection) (p13:sp5 ph5):f2 ; ----------------- CO --> CA d15 ; 1/4J(CA,C') (p14:sp3 ph0):f2 d7 (p5 ph0 3u p6 ph8 3u p5 ph0):f3 (p14:sp14 ph0):f2 d23 pl13:f3 (p14:sp3 ph0):f2 ; BSP 3u (p13:sp6 ph0):f2 3u cpds1:f1 ; 1H decoupling on 3u cpds3:f3 ; 15N decoupling on (p13:sp1 ph7):f2 ; refocus CO/CA, chemical shift evolution CA 3u (p14:sp14 ph0):f2 d20 ; dd20 IN20=1/2SWH (p12:sp12 ph15):f2 ; 1.25ms RE-BURP.256 CA selective 180 pulse 3u (p14:sp14 ph0):f2 d0 ; id0 IN0=1/2SWH d5 3u do:f1 ; 1H decoupling off p16:gp2*EA d18 pl1:f1 (p13:sp2 ph17):f2 d8 do:f3 ; 15N decoupling off (p1 ph0):f1 ; ----------------- CA --> HA d24 (p14:sp3 ph17):f2 d9 (p2 ph0):f1 d24 (p13:sp1 ph14):f2 (p1 ph8):f1 d4 (p14:sp3 ph17):f2 (CEN_HC1 p2 ph0):f1 d4 (p1 ph0):f1 d29 (p2 ph0):f1 p19:gp3 300u pl12:f2 50u BLKGRAD go=2 ph31 cpds2:f2 d1 do:f2 wr #0 if #0 zd 3m ip14 3m ip14 igrad EA lo to 3 times 2 ; E/AE selection 5m ip14*2 5m ip17*2 5m ip31 5m ip31 5m id0 ; IN0=1/2SWH for 13CA(i-1) 5m dd20 ; IN20=IN0 (>d20/l0) lo to 4 times l0 ; 13CA(i-1) 5m rd0 ip3 5m rd20 ip13 lo to 5 times 2 ; States-TPPI 5m id10 ; IN10=1/2SWH (13CA/CD) 5m dd30 ; IN30=IN10 (>d30/l10) lo to 6 times l10 ; 13CA/CD(i) exit ph0= 0 ph2= 1 ph3= 0 ph13=0 ph9= 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 ph4= 0 2 ph5= 0 0 2 2 ph14=3 ; E/AE selection ph15=0 0 0 0 2 2 2 2 1 1 1 1 3 3 3 3 ph17=0 ph7= 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ph8= 1 ph31=0 2 2 0 2 0 0 2 2 0 0 2 0 2 2 0 2 0 0 2 0 2 2 0 0 2 2 0 2 0 0 2 ;p16 gradient E/AE 13C ;p19 p19=p16/4 gradient E/AE 1H ;d16 200u gradient recovery ;gpz0 -80 ;gpz1 70 ;gpz2 90 ;gpz3 90.3 ;l0 32 (<35 !!) ;l10 64 (<66 !!) ;cnst22: CD/CA chemical shift (offset, in ppm) [56ppm, CD/CA selection] ;cnst23: CD/CA chemical shift (offset, in ppm) [59ppm, CA selection] ;pl2: 0W, 1000dB ;spnam1: 13CA/13CD sel. pulse 90 deg [Q5.1000, 320us, on res] ;spnam2: 13CA/13CD sel. pulse 90 deg [Q5.1000, 320us, on res] ;spnam3: 13CA/13CD sel. pulse 180 deg [Q3.1000, 256us, on res] ;spnam12: 13CA/13CD sel. pulse 180 deg [Q3.1000, 1000us, on res] ;spnam4: 13CO sel. pulse 180 deg [Q3.1000, 256us, off res] ;spnam5: 13CO sel. pulse 90 deg [Q5.1000, 320us, off res] ;spnam6: 13CO sel. pulse 90 deg [Q5tr.1000, 320us, off res] ;sp1: 13CA/13CD sel. pulse power 90 deg [Q5.1000, p13=320us, on res] ;sp2: 13CA/13CD sel. pulse power 90 deg [Q5.1000, p13=320us, on res] ;sp3: 13CA/13CD sel. pulse power 180 deg [Q3.1000, p14=256us, on res] ;sp12: 13CA/13CD sel. pulse power 180 deg [Q3.1000, p12=1000us, on res] ;sp4: 13CO sel. pulse power 180 deg [Q3.1000, p14=256us, off res] ;sp5: 13CO sel. pulse power 90 deg [Q5.1000, p13=320us, off res] ;sp6: 13CO sel. pulse power 90 deg [Q5tr.1000, p13=320us, off res] ;p12: pulse length 13CA/13CD sel. 180 deg [Q3.1000, 1000us] ;p13: pulse length 13CA/13CD/13CO sel. 90 deg [Q5.1000, 320us] ;p14: pulse length 13CA/13CD/13CO sel. 180 deg [Q3.1000, 256us]