Documents
Resources
Learning Center
Upload
Plans & pricing Sign in
Sign Out

Checking 90o pulses of 15N_ 13C and 1H

VIEWS: 33 PAGES: 13

									Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe NOTE: It is understood that you would have had prior training on at least one of the instruments before using this manual for setting up the instrument for protein data collection. Also it is understood that you would have had a trained super-user walk through the procedure before you are sent out on your own. NOTE: These procedures will change settings in the edprosol, which is the brain of Bruker software. Changing these parameters will affect the entire system and every experiment ran after making changes to edprosol. NOTE: When changing settings for any nucleus (1 H/13C/15N) in edprosol use the solvent setting All. While the 15N Urea sample is in DMSO the 2D/3D protein experiments are typically ran with the D2O solvent setting. When typing gpro to port in standard parameters from edprosol the values stored in edprosol under a specific solvent will only be ported into an experiment if that same specific solvent is selected in the experiment (i.e. if the DMSO was selected in edprosol when updating the optimized 90º 15N pulses and you want to run the CBCANH with the solvent setting D2O, running gpro WILL NOT update the current optimized parameters for the 15N signal because it was updated in edprosol under the wrong solvent ). It is easier to just select All solvents in edprosol for each nucleus rather than select a specific solvent and being left wondering why you parameters are not updating when you type gpro. Also the correct probe needs to be selected but that will only be done by a trained super-user, if the 5mm PATXI 1H-13C/15N XYZ-GRD Z8562/0007 [32] probe is not selected tell someone that will change it for you. NOTE: 1H 90º pulses must be optimized using the sample of interest therefore tuning must also be done on sample of interest. Optimizing the 90 pulse widths for 1H MUST be done before collecting any 2D or 3D protein data set. However, if the 13C/15N pulses were recently updated you can skip these steps and go directly to updating 1 H pulses. NOTE: Pulse power levels are sample dependent and very sensitive to the amount of salt in the solution. Always use the lowest salt concentration needed to keep the protein soluble. NOTE: These are the settings that need to be used for edprosol to work correctly. Before updating any pulse length/power, make sure these are set for the correct nucleus Table 1: edprosol setup routing channels. Probe 1H-13C/15N XYZ-GRD Z8562/0007 [32] 1H-13C/15N XYZ-GRD Z8562/0007 [32] 1H-13C/15N XYZ-GRD Z8562/0007 [32]

Solvent All All All

Nucleus 13 C 1 H 15 N

Logic channel F1+F2 F1+F2 F3

Amp channel A1 A2 A6

NOTE: Even though gpro does work if all the settings are correct, it is still a very good habit to check that your parameters are correct before pressing zg and walking away from the instrument. An extra hour spent setting up an experiment can save you 3+days of data collection MAKE SURE YOUR EXPERIMENTS ARE SET UP CORRECTLY THE FIRST TIME!!!!!! If you have any questions ask someone that would know.

1

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe

Tuning and 90o pulse calibration of 15N
Tuning the probe: 1. With airlift on , place the standard 15N-labeled urea sample in the magnet – turn off air lift 2. Lock on DMSO (NOTE: for DMSO type >edte and change set temperature to 308K) 3. Type >rsh TXI-DMSO to recall standard DMSO shim set 4. Manually touch up shims Z1 and Z2 5. Disconnect only the high-pass 13C/2H filter from broadband preamp (1B) (second from top on front of pre-amp) and replace with low-pass 15N/2H filter (NOTE: The 15N/2H filter should be disconnected from the Amp 6 cable. When you hold the filter in your hand there should still be a cable running to the 15N channel on the probe. Make sure the cables are only finger tight) 6. Click on File -> Open -> Data Sets By Name... and open standard file 15N_TUNE 7. Type >edasp to bring up preamp routing map (NOTE: this is only for tuning and the screen should look like figure 1) Save and Exit back to main window Figure 1: Channel/SGU/AMP/Preamp Routing Map (NOTE: For
15

N Tuning only!!!)

8. Type >wobb and press Enter 9. Type >a (or >acqu) and press enter (NOTE: this brings you to an acquisition window)

2

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe 10. Using the RED match and tune sticks, adjust signal so that the tip is matched to 50ohms and centered on nucleus frequency (NOTE: see figure 6 for example, this is for 13C signal but will give an idea of what a tuned/matched signal looks like) 11. Change the wobb frequency to 1H by clicking on wobb-sw and answer yes 12. Tune 1H using the GOLD match and tuning sticks (same method as step #11) 13. Once matched and tuned on both 15N and 1H, type >stop to stop wobb acquisition. 14. Re-cable back to original configuration by disconnecting the low-pass 15N/2 H filter from the broadband preamp (1B) and reconnecting the high-pass13C/2H filter (NOTE: make sure to reconnect low-pass 15N/2 H filter directly to Amp 6) 90o Pulse Calibration (NOTE: This is done after the probe has been tuned to 15 N) 1. The 1H spectrum needs to be observed before decoupling 15N to observe the anti-phase doublet which appears around 5.5ppm 2. Open the previous 15N decouple file by clicking on File -> Open -> Data Sets By Name... and open standard file 15N_DECP90###### (NOTE: the last set of numbers is a date reference, open file with newest date) 3. Make sure correct pulse program loads, type >pulprog and press enter (NOTE: the correct program is decp90f3 if the correct program loads simply press enter again if not type in the correct program and press enter then type >gpro and press enter) 4. After data set is open type >new and rename using current date (NOTE: this will port over all of the parameters into the new file) - Save and Exit to main screen 5. Now Type >edasp to bring up preamp routing map (NOTE: This screen is different from the tuning screen and it should look like figure 2. If it does not look like figure 2 turn on the appropriate settings by clicking on them) -Save and Exit back to main window 6. Type >gpro and press enter to make sure correct settings are entered

3

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe Figure 2: Standard Channel/SGU/AMP/Preamp Routing Map (NOTE: for Protein Structure Data

Collection)

7. Type >ased and press enter - this brings up an acquisition parameter set window 8. Scroll down to PL3 - the power level for a pulse on F3, in this case nitrogen 9. Set power level for the 15N pulse (PL3) to 120dB which will turn off the decoupling pulse (NOTE: -6.0dB is max power and 120dB is no power - NEVER USE MAX POWER!!!) Save and Exit back to main screen 10. Type >rga to adjust receiver gain 11. Type >rg to check receiver gain (NOTE: receiver gain<100 is good) 12. Type >ns and set number of scans to 1 then type >ds and set number of dummy scans to 0 13. Type >zg (zero go) press enter to run 14. After acquisition, type >efp, zoom in on anti-phase region (~4.5-6.0ppm) and then phase correct manually (NOTE: one peak should be pure (+) and one should be pure (-), it doesn’t matter which peak is which, just make sure that you remember the order see Figure 3)

4

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe Figure 3: Non-decoupled 15N spectrum of 15N Urea, notice anti-phase region near 5.5ppm

15. Type >iexpno to create/advance to the second sub-directory (NOTE: Directory 1 is for nondecoupled spectrum; Directory 2 is for decoupled spectrum. This allows you to compare the two spectra to ensure you are getting decoupling.) 16. Under directory #2 click on utilities and then set O1 to the center of the anti-phase doublet. To do this simply place cursor in center of doublet and press center mouse button (NOTE: typically this should be about 3275.0Hz) 17. Return to main screen by clicking on return and type >gpro and press enter. gpro, or getprosol calls up the saved pulse parameters from edprosol and enters them into the 15N-DECP90YOURDATE file. 18. Type >zg again. 19. After acquisition, type >efp, this time the anti-phase peaks should be a null or very close. If they are not, adjust PL3 slightly and re-collect the spectrum. Repeat until the peaks reach a null. See Figure 4 (NOTE: Do not adjust the power to -6 dB! If you need to change the setting more than +/0.2db, something may be wrong with either your setup or the hardware, and either Joe or Sara should be contacted. The typical value for PL3 is -3.2db) 20. You can now compare the decoupled spectrum to the non-decoupled spectrum by typing >re 1 which will take you back to the first directory

5

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe Figure 4: Decoupled 15N Urea Spectrum, notice that the anti-phase region is much lower in intensity than the non-decoupled spectrum figure 3

21. If PL3 value was changed, type >edprosol and enter your new value for 15N, routed through F3 using Amp 6. The standard hard pulses should open first and enter new PL3 value in edprosol in the box under power level for P90. Make sure to recalculate all the saved pulses, including any Standard Soft Pulses. Click Save and answer any questions by clicking on “calc.” for each box that is a number other than 120db. See figure 5 for example. (NOTE: use the solvent setting All. While the 15N Urea sample is in DMSO the 2D/3D experiments are ran with the D 2O solvent setting. When typing gpro to port in standard parameters from edprosol the values stored in edprosol under a specific solvent will only be ported over into an experiment if that same specific solvent is selected in the experiment. It is easier to just select All for solvents in edprosol rather than select a specific solvent because this will port over the correct parameters into any experiment regardless to what solvent is selected)

6

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe Figure 5: Edprosol window for entering 15N decouple pulse power (NOTE: new PL3 value is entered in the box labeled P90 Power level)

22. Eject Sample and set temperature back to 298Kin edte

*****Set temperature back to 298 K before placing protein sample in magnet*****

7

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe

Tuning and 90o pulse calibration of 13C

NOTE: The steps for tuning and decoupling 13C are very similar to the tuning and decoupling 15N procedure. You do not need to do any re-cabling or change the pre-amp route map for 13C sample.

Tuning the probe 1. With the air lift on, place standard 13C-labeled alanine sample in the magnet – turn off air lift 2. Lock on D2O 3. Type >rsh TXI-D2O to recall standard D2O shim set 4. Manually touch up shims Z1 and Z2 5. Open the latest 13C-DECP90-#### pulse file. Change the file name by typing >new and changing the date code on the filename to reflect the current date. (NOTE: the last set of numbers is a date reference, open file with newest date) 6. Make sure correct pulse program loads, type >pulprog and press enter (NOTE: the correct program is decp90 if the correct program loads simply press enter again if not type in the correct program and press enter then type >gpro and press enter) 7. Type >wobb and press enter 8. Type >a (or >acqu) and press enter (NOTE: this brings you to an acquisition window) 9. Using the BLUE match and tune sticks adjust signal so that tip is matched to 50ohms and centered on the 13C frequency Figure 6: Correctly tuned and matched 13 C signal

8

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe 10. Change wobb frequency to 1 H by clicking on wobb-sw and answer yes 11. Tune 1H using the GOLD match and tuning sticks (same as step #8) 12. Once matched and tuned on both 13C and 1H type stop to stop wobb acquisition 90o Pulse Calibration 1. First the 1H needs to be observed without decoupling 13C to see the anti-phase doublet of doublets 2. Since the pulse program is already open you do not have to open it again, so after tuning, type >ased and press enter - this brings up the acquisition parameter set windows 3. Scroll down to PL2 which is the power level for a 90 degree pulse on F2. 4. Set power level to 120db which will turn off decoupling pulse (NOTE: -6.0db is full power and 120db is no power NEVER USE MAX POWER!!!) Save and Exit back to main screen 5. Type >rga - this will show you the receiver gain (NOTE: receiver gain<100 is good) 6. Type >zg to run one pulse 7. After acquisition, type >efp, zoom in on anti-phase region and then phase correct manually (NOTE: make sure that you remember the order of the peaks, one set should be pure (+) and one set should be pure (-). The peaks are anti-phase with no decouple pulse due to the length of the relaxation delay being equal to 1/2J; this delay causes anti-phase magnetization of the spins. When the 90 is reached, the magnetization is purely from multiple quantum coherence so the peaks disappear. ) 8. Type >iexpno to create/advance to the second sub-directory (NOTE: Directory 1 is for nondecoupled spectrum; Directory 2 is for decoupled spectrum. This allows you to compare the two spectra to ensure you are getting decoupling.) 9. Under directory #2 click on utilities and then set O1 to the center of the anti-phase doublet. To do this simply place cursor in center of doublet and press center mouse button (NOTE: typically this should be about 811Hz) 10. Return to main screen and type gpro 11. Type >zg again 12. After acquisition, type >efp. This time the peaks should be a null. If they are not, adjust PL2 slightly until the peaks reach a null. (NOTE: Do not adjust the power to -6 dB! If you need to change the setting more than +/- 0.2db, something may be wrong with either your setup or the hardware, and either Joe or Sara should be contacted. The typical value for PL2 is -2.6db)

9

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe 23. If PL2 value was changed, type >edprosol and enter your new value for 13C, routed through F1+F2 using Amp 1. The standard hard pulses should open first and enter new PL2 value in edprosol in the box under power level for P90. Make sure to recalculate all the saved pulses, including any Standard Soft Pulses. Click Save and answer any questions by clicking on “calc.” for each box that is a number other than 120db. See figure 7 for example Figure 7: Edprosol for 13C (NOTE: calculate pulse lengths for both F1 and F2 channels with A1 amplifier for 13 C, a couple of the protein experiments use both channels)

10

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe

Tuning and 90o pulse calibration of 1H
NOTE: 1H 90º pulses must be optimized using the sample of interest therefore tuning must also be done on sample of interest. This MUST be done before collecting any 2D or 3D protein data set, however if the 13C/15N pulses were recently updated you can skip these steps. Update 13C/15N pulses every 3-4weeks

Tuning the Probe 1. With the air lift on, place your protein sample in the magnet – turn air lift off 2. Lock on the deuterated solvent (NOTE: For proteins use D2O) 3. Type >rsh TXI-D2O to recall standard D2O shim set 4. Type >gradshim and press enter to open the gradient shim protocol. Select 1D and start gradshim, run 1D gradshim three times and 3D gradshim once. (NOTE: 1D gradshim is used for mostly ~90% H2O samples and 10%D2O while 1D2H gradshim is used for mostly ~90%D2O samples and 10%H2O, 3D gradshim adjusts spin and non-spin shims) 5. Follow steps for
15

Nitrogen and 13Carbon tuning but do not calibrate

90o Pulse Calibration 1. Type >new and make a new file for your sample. 2. After sample is tuned type >1h and press enter to recall the standard 1D proton pulse parameters. (NOTE: this pulls in the zg30 pulse program do not use this pulse program to calculate the 1H 90. The tip angle is only 30 and will give false maximum 90) 3. To calculate the 1H 90 hard pulse you must type >pulprog and set pulse program to zg (NOTE: the pulse program zg must be used to calculate 1H 90. The tip angle is the full 90 and places maximum signal in the receiver coils) 4. Type >ns and press enter to set number of scans to 1 then type >ds and press enter to set number of dummy scans to 0 5. Type >rga to adjust receiver gain 6. Type >rg to check receiver gain (NOTE: receiver gain of 4 or less is good, low receiver gain indicates large signal, if you have a large rg with a primarily H 2O sample something is wrong) 7. Type P1 and press enter –set the pulse length well below your 90 pulse somewhere around 3µs should be good 8. Run sample by typing >zg 9. Process the FID once acquisition is finished by typing >efp press enter then type >apk press enter then type >abs and press enter (NOTE: check for a clean, phased water peak ~4.6ppm)

11

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe 10. Type >paropt –press enter select P1 -press enter then enter the pulse length at which to begin press enter, then enter the value to increment -press enter and finally the total number of experiments -press enter to run (NOTE: typically start at 5μs, increment by 5μs and run 12 experiments. This will take you through a pulse length of 60μs which should be more than plenty to find the 360 null) 11. With paropt, you look for a 360 null, this means the signal has been pulsed through 360 degrees and is now aligned back to the positive z-axis. We look for the 360 because measuring the 90 directly could give false results due to saturation transfer and signal dampening of the H 2O peak. There should be two signal nulls, one at 180 and one at 360, by using paropt and starting at a sufficiently low pulse length we can see both nulls; the 360 will be lower in intensity than the 180. However, the 180 may not give a proper null because it might be out of phase. Notice the 360 null signal is minimized and there is a region with equal area of (+) and (-). When the 360 null is found write down this pulse in microseconds and divide by 4 to give an optimized 90º pulse. (NOTE: For the 600MHz instrument the 360º null is typically around 30-40μs. In figure 8 the 360 null is at 31us so to find the 90º simply divide by 4 and get a 90 of 7.75μs) (NOTE: paropt creates a subdirectory with the main file named 999, if you want to run paropt more than once you must first be back in the main directory to restart paropt. To do this type >re file# 1) 12. After running a rough paropt with p1~5us it is beneficial to re-run paropt to “hone in” on the correct 360. To do this simply type >re file# 1 to restart paropt from the first sub-file in the directory then type >paropt and press enter again. This time start closer to the 360 and increment at smaller intervals (i.e. if the first paropt with 5μs increments found the 360 at 31μs run paropt again starting from 30μs and incrementing by 0.5μs or 1μs) Figure 8: 1H Paropt for pulse length (P1) (NOTE: Start P1=5μs increment by 5μs for 8 experiments to pass through 40μs, the 360 null is in between 30μs and 35)

12

Guide for Instrument Setup and Pulse Calibration on the 600MHz Spectrometer with TXI Probe 13. Enter new PL1 value into edprosol and calculate pulses. Type >edprosol and enter your new value for 1H, routed through F1+F2 using Amp 2. The previous hard pulses should open first and enter new PL2 value in edprosol in the box under power level for P90. Make sure to recalculate all the saved pulses, including any Standard Soft Pulses. Click Save and answer any questions by clicking on “calc.” for each box that is a number other than 120db. Figure 9: Edprosol for 1H (NOTE: calculate pulse lengths for both F1 and F2 channels with A2 amplifier for 1H, a couple of the protein experiments uses both channels)

13


								
To top