VnmrJ 2.1B : How to use for beginners(2) Shin Jae Sun KAIST whoa@kaist.ac.kr login Auxiliary computer NMR computer (OS : linux) Laser Printer (OS : WinXP or Linux) NMR Monitor (you will work here) • Login • ID, passwd • logout if someone already has logged in, then log in • If common id is in use, then just use. 1. You will see this page after login. 2. click right button of mouse to open terminal 3. Type “vnmrj “ on terminal to run vnmrj Or just click the icon or quick launch Menu Command line jexp2 Experiment library Double click ‘exp2’ or type “jexp2” Spectra display screen if you want to begin experiment on exp2 (check the assigned number range for person) If you want to make new experiment {ex)exp7}, type “cexp(7)” Click “Spin&temp” on ‘Setup’ tab Tab for experiment Text display screen Sample environment status Experiment status Message / Log temp Or type “temp” at command line Alternative way to control temperature How to control temperature 2. click “Regulate Temperature at this Value” 1. Type ‘temperature’ or drag the bar as you want Drag the bar as you want, then close the window Wait till temperature is changed as you want Then, Insert a NMR tube NMR cell Normal Shigemi Shigemi (bruker) (Varian) Fit sample to rectangle e click “ Eject Sample” or type “e” on command line Sample injection insert here 1. Remove all the material which have metal and magnetic property such as credit card, watch, coin and etc... 2. Check the previous NMR tube is present on magnet before inserting your NMR tube i 1. Click “ insert Sample” or type “i” on command line 2, Wait for 2~5 minutes for temperature 3, Click “Lock” on ‘Setup’ tab lock tn=‘H1’ dn=‘C13’ dn2=‘N15’ su Tuning 1. type “tn=‘H1’ dn=‘C13’ dn2=‘N15’ su” on command line 2. then now go to tune... Lock automatically 1. Click “Find Lock” or type “lock” on command line 2. If autolock finishes, status returns “idle” and ‘activiate Lock’ is checked Tuning unscrew ‘right below cable’ & connect to below of LED for H1 •ATTN : always 9 •CHAN : H1 – 1 •Adjust LED value till ~001 by tuning rod •tuning off LED by set CHAN as 0 •ATTN : always 9 •CHAN : C13 – 2, N15 - 3 •Adjust LED value by tuning rod : C13 ~ 001, N15 ~ 004 •tuning off LED by set CHAN as 0 H1 C13 N15 Tuning rod unscrew ‘C13 cable’ & connect to below of LED unscrew ‘N15 cable’ & connect to below of LED Automatic gradient shimming Click “Setup Gradient Shimming” v Automatic gradient shimming 1. Check “Gradient Shim” on ‘Acquire’ tab 2. Click “Gradient Autoshim on Z” 3. If shim is well done, click “Quit Gradient Autoshim” to return Manual shimming Click “Shim” on ‘Setup’ tab v Manual shimming - 1. Change lock amplitude by increasing ‘lock gain or power’ till 80~90 2. Change Z axis shim till lock amplitude reaches maximum 3. Change X,Y axis shim till lock amplitude reaches maximum 4. If lock amplitude reaches 100, reduce the lock gain a little 5. Repeat ‘step 1,2’ till no more increase at lock amplitude 6. If shimming is finished, reduce a lock amplitude till 45~55 or auto “lock” again + scale v Check shim is proper by presaturation method • Type “water” on command line • Type as : satmode=‘nnn’ pw=1 ss=0 sw=6000 np=8192 tpwr=58 ga • Type as : f full aph # • You could do same action using VNMRJ GUI by clicking “Acqure” tab. But in this presentation, I suggest to use command for easy explanation. Additionally.. • satmode=‘nnn’ : turn off presaturation, ga : show acquired spectrum after doing experiment • aph : auto phase correction, f : full spectrum, full : full screen v 1. Peaks must be sharp and symmetric. If peak is not good shape, do shimming again 2. Click redline button nl : Position cursor at the nearest line Measure tof movetof : move transmitter offset 3. Set redline near the center of peak & type “nl” 4. type “movetof” & look information for ‘tof” value 90 pulse calibration • Type as : array(‘pw’,20,36,1) • Check the array list by click “Textout” on ‘Setup’ or ‘Acquire’ tab array ( ‘parmeter’. step number, starting value, increment) 90 pulse calibration • Type “ga” to do experiment • When negative peak turns positive peak, type “aa” • type “dssh” and “dssl” aa : abort experiment dssh : display stacked spectra horizontally dssl : label a display of stacked spectra 90 pulse calibration (for example) • Change ‘starting value’ as pw[7](or pw[8] as you want) • Reduce ‘increment’ as 0.2 (or 0.1) • ex) array(‘pw’,47,20,0.2) or array(‘pw’,pw[7],20,0.2) • Type “ga” 90 pulse calibration (for example) 1. Select the peak which seems to be null (in this case : peak 7) 2. Type as : pw=pw[7]/4 or pw=48.2/4 • 3. Divide pw[7] by 4 for 90 degree Remember ‘tof’ and ‘pw’ value for further experiments • type “tof?” & “pw?” : check parameter value HSQC • Heteronuclear Single Quantum Coherence spectra • Detection of amide nitrogen-proton cross peaks. • Easy and Sensitive • Usually used by titration for many purpose Acquire 15N-HSQC spectra 1. Move to experiment number where you want to use • 2. ex) jexp113 : in my case exp113. Check your assigned regtion Type “gNhsqc” on command line to bring 15N-HSQC pulse sequence Before acquiring 15N-HSQC spectra 1. Type “man(seqfil)” or click “Sequence Manual” : Display manual 2. Step for 15N-HSQC 1. 1D spectra of 15N-HSQC • 2. simple 2D spectra • 3. nt : scan number, sw : sweepwidth of H1 dof2 : offset of N15, sw1 : sweepwidht of Y axis(N15) Obtain 2D spectria 1D 15N-HSQC spectra 1. Input ‘pw’ and ‘tof’ which are measured before ex) pw=12 tof=-225.3 2. Type as : 3. 1. dpwr2=44 dmf2=1/272e-6 : 15N decoupling 2. sw=9000 np=1024 ss=4 tpwr=58 d1=1 nt=4 gain=24 3. setoffset(‘N15’,120):dof2 : set N15 offset on 120ppm 4. ni=1 phase=1 : for obtaining 1D spectra 5. C13refoc=‘y’ : for 13C,15N labeled protein. unless ‘n’ instead of ‘y’ 6. NHonly=‘n’ NH2only=‘n’ : full spectra for NH, NH2 Type “ga” Select 1 guide line or 2 region select line Zoom in(+) or out(-) Integration Display scale bar : ‘dscale’ Grab & move Show threshold Phase correction Refresh Return to above option Phase Correction 4. Adjust peak shape properly 1. Click here for ‘Phase correction’ 5. Click here to finish ‘Phase correction’ 2. Make baseline flat on left 3. Make baseline flat on right 1. type as : setref dscale. setref : set frequency reference 2. Check 1D spectra dscale : display scale below spectrum 3. 1. If spectra is good enough, then ‘nt=4’. (conc. > 0.4mM) 2. Unless type ‘nt=8’ or set nt as 4*N [N: integer] and re-check Check the sweepwidth 1. If peaks don’t appear above 10.5ppm, sw=7500.(recommended) 2. If peaks don’t appear above 9ppm, sw=6000. (in case of no TRP) 4. Type as : sw1=2200 ni=32 phase=1,2 f1180=‘y’ ss=32 : for 2D spectra 5. Type “go” : I don’t suggest “ga” for it bothers you during acquisition by displaying 1D spectra continuously Select cross guide lines or region select grid Zoom in / out Trace (1D slice) Projection Refresh Rotate Scale +20% Scale -20% Phase2D Peak Picking Return to above option 1. Type as : 1. setref setref1(‘N15’) : for reference 2. sp=5.5p wp=5.5p : select display region 3. wc=150 wc2=150 : adjust window size 4. wft2da : display 2D spectra 2. If peaks are too strong or too weak, adjust magnitude by click ‘Scale +/- 20% button’ 3. Measure sweepwidth of N15 1. 2. Check the boundary 1. downfield : 131.5ppm. 132ppm 2. upfield : 105.37ppm 105ppm Calculate sweepwidth 1. Δ=132-105=27ppm 2. sw=27*dfrq2=~1641 3. Type “sw=1700” (~28ppm) 4. N15 offset : 132-13.5 =118.5ppm 5. Type “setoffset(‘N15’,118.5):dof2” 6. Type “f1180=‘n’” : folding option is off :dfrqw=60.781 Determine resolution • Check the spectrum : ni=32 for sw=1700 • Titration, Relaxation : ni=128~192 for sw=1700 • High Quality spectrum : ni=192, 256 for sw=1700 Plot 1. Type “dconi(‘dpcon’,12,1.3)” : show contour (12 level, 1.3 factor) 2. Type as : 3. 1. setref setref1(‘N15’) sp=5.5p wp=5.5p wc=150 wc2=150 2. setLP1 gaussian wft2da : plot after linear prediction and weigh function Plot •ppa : plot a parameter list •plot2D : plot 2D spectra • pos : positive • levle : 12, factor : 1.3 •page : submit plot to printer or file 1. ppa plot2D(‘pos’,12,1.3) page : print on A4 paper 2. ppa plot2D(‘pos’,12,1.3) page(‘a.ps’) : print as PS file(a.ps) Save 1. cd(userdir+’/data/yourname’) : Change directory 1. 2. ex) cd(userdir+’/data/whoa’) svf(‘filename’) : save fid 1. ex) svf(‘20081019_XXX_gNhsqc_25C_Titration’) Compression 1. Transfer compressed file by SSH2 (linux) or SSH Secureshell Transfer(WinXP) 2. Process with NMRPipe Next.... • How to process FID with NMRPipe • How to convert • FID(VnmrJ) pipe(NMRPipe) ucsf(Sparky)