Guide to the 600 and 800 Note means enter key

Transcription

1 Guide to the 600 and 800 Note means enter key 1. Check that no one else is already using the spectrometer! a. Is the Fid Flash panel flashing? b. Does the Acquisition Information panel says no acquisition running? c. Are there no queued items in the Spooler panel? 2. Check that there is a green cold light on the cryoplatform. 3. To take a sample out of the magnet or to put in yours, click B tab at the top of the main Topspin window or type bsmsdisp to make the BSMS panel visible then LIFT button (it will become green when active). If the previous user s sample is in the magnet, put it in the fridge (or wherever else their instructions specify). Hit the LIFT button again (it will become grey) to lower the sample into the magnet or whenever LIFT is not required (when idle). 4. If you want to record your experiments NOT at 298K (note VTU panel always displays current temp.), then you will have to change the temperature: a. Click T tab or type edte to bring up temperature control module and go to Config tab, load in the configuration file that matches closest to your desired temperature. File>load configuration e.g. tci_670l_285kk_corr.tcf (for 600) or tci_800l_275k_corr.tcf (for 800). b. Change the temperature to what you want. Go to Main display tab, then Change button (next to Target temp. box). Make sure you change the Sample Target Temp and not the Sensor temperature. c. Avoid large temperature jumps (>20 K) in one go as it is bad for the sample and the probe. If making large temperature changes and you don t want to do this manually in steps, use the Ramp function. Click Ramp tab, then set Ramp target temp and ramp rate (should be no more then 2K/min). If the desired temperature is lower than 293 K, you will need extra cooling: d. Switch on (orange switch) the cooler box (the BCU-05) behind the 600 magnet or next to the console (on 800). 5. The default temperature configuration file is tci_800l_300k_corr.tcf (for 600) and tci_800l_298 so please change back to this when you are finished (and turn BCU- 05 off for 600). 6. To change the sample temperature, click Main display tab, then Change button. 7. If the temperature you want is more than 10 o from the current T (either way), change it using a ramp: click T tab>ramp with a setting of ~1.5 o /min. It is also important NOT to use the BCU-05 at temperatures higher than 293 K on the 600 only. 8. Once the temperature has stabilized, get your sample in there according to 3 above (best not to put it in too early if you are making a big T change sometimes it can overshoot quite a bit). 9. Lock with lock then select the H2O+D2O or D2O option according to your sample. 10. Read in an existing experiment which has all the channels ( 1 H and/or 15 N and/or 13 C) you will be using for this sample (e.g. 15N-HSQC, triple resonance), then type edc to copy into a folder with your name/date. Type atma for automatic tuning and matching.

2 11. Type topshim to do automatic shimming. 12. If the lock signal is no longer visible in the lock display window, press gain (in LOCK section of the BSMS panel to access click B tab or type bsmsdisp) and scroll mouse wheel up or down until the signal sits at the top or 2 nd topmost grid lines. 13. Press phase in the AUTO section of the BSMS panel (if invisible, click B tab or type bsmsdisp). 14. Manually shim/check the off-axis shims using BSMS panel if desired. 15. Open the first experiment you want to run by double clicking on the experiment in the browser window. Check the experiment number tab pressed has actually loaded the experiment you intend to run. 16. Type pulsecal to measure the 90 o for 1 H. Record this. This program will automatically update your current experiment with the new pulse length. Make sure the 90 o pulse is at least 8 µs long. For complicated experiments (e.g. triple resonance type), it is best to use a 90 o pulse of at least 10 µs. Use calpowlev to determine how much you will need to drop the power level by with the 10 µs pulse. 17. To obtain power levels for different pulses, use the prosol table. You can get it to read all relevant parameters into your current pulse program using getprosol 1H pulse_length pulse_power (e.g. getprosol 1H ), where pulsecal gives 1 H pulse of 8.5 µs at -7.9 db. This will read in values for all pulses, not just proton ones. You should then check that all of the numbers look sensible. A list of commonly used experiments is kept in the /stanexp folder. Experiments in this list have been checked and should have the correct parameters, except for pulse power/length which will be updated with getprosol. In most cases, you should not need to change parameters apart from: ns, TD, SW, O1P, O2P, O3P (depending on experiments). 18. To start your experiment, type zg 19. You can queue experiment by typing zg in the experiments you want to run sequentially. 20. To process 1D data, the command is gfp (stands for Fourier transform using Gaussian window function and apply phase correction). For 2D data, the processing command is xfb. General note: To set up your experiments, you must ensure that you DON T USE EXCESSIVE POWER LEVELS OR ANY PULSEPROGRAMS THAT USE TRIM PULSES. Easiest would be to copy a recent experiment or one from the /stanexp directory. The power levels are posted on the board and are also in the cryoprobe power guidelines manual (blue and red folders) next to the computer. Consult Joel or Ann before setting up anything that you are not 100% sure about. If we must use a pp that contains a trim/purge pulse, we need to set it to be 2 db lower than the hard pulse (which is 5.1 db on 600 and 3 db on 800 for the moment). Keep an eye on the cryoplatform. If any light OTHER than the green cold light appears it is essential that you stop your experiment and consult Ann.

3 Comments on the cryosystem 1. It is VERY expensive if we do any damage to it, we are looking at a minimum of 10-20K to fix and several months in Switzerland (for the probe, not for you!). 2. You should essentially not have to worry about the cryosystem in your experimental setup, except to check that the lights on the two boxes are green and solid. If they aren t, talk to Ann, Joel and Bill. 3. The sample depth is 21 mm with this probe. Use the fixed depth gauge that is shaped like a rectangular prism (not the round one). 4. Temperature limits for cryoprobe are 0 50 degrees. 5. There are some limitations in pulse powers and durations. It is essential that we adhere to these (see tip 1): a. 1 H pulse power for hard pulse must be no stronger than 5.1 db on 600 and 3 db on 800. b. For 1 H TOCSY, you can go up to 200 ms mixing time (much longer than we use) with whatever pulse level you get for a 29 µs 90 o. A 30-µs 90 pulse will be ~14 db should be fine to go up to ~100 ms mixing time at this power level. c. For 15 N decoupling, 140 ms is the maximum acquisition time you can have (corresponds to 2K points and an SW of ~13.5 ppm) if you use a 170 µs decoupling pulse. I recommend using a 250 µs pulse (and GARP4), which should allow you a bit of leeway in acquisition time. d. Rule of thumb: the % by which you increase the 90 pulse time = the % by which you can increase acquisition time. e. Note that if a pulse sequence contains simultaneous 15N and 13C pulses, then the power of each pulse must be reduced by 3 db and the pulse times recalibrated. In Topspin2.1, all PP's have been re-written to remove simultaneous hard pulses. There are limits to CC TOCSY and 15N spinlocks too. Check with Ann or Joel if not sure not worth the risk!

4 Experiments available in stanexp directory: Theses experiments have been checked so all you need to do is an edc, then a pulsecal and getprosol to have correct pulsepowers and pulse lengths. Parameters you may want to edit are: ns, SW, O1P, O2P and/or O3P. The stanexp dir contains acquisition and processing parameters as well as raw and processed data so you get a preview for what the experiment is like. To stop this dir getting accidentally written over, it is write protected so you will have to do an edc before you can set up or run the experiment. Experiments shown in grey are yet to be implemented. Unlabeled samples: stanexp/1 zg; 1D with no water suppression stanexp/2 zgpr; 1D with pre-saturation stanexp/3 p3919gp; 1D with Watergate (gradient) stanexp/4 dipsi2gpph19; 2D 1 H TOCSY (mixing time 35 to 70 ms) stanexp/5 noesygpph19; 2D 1 H NOESY (mixing time 50 to 400 ms) stanexp/6 cosydfgpph19; 2D 1 H COSY stanexp/7 roesydfgpph19; 2D 1 H ROESY (for very small molecules <~3kDa) 15N-labeled samples: stanexp/11 hsqcetf3gpsi; 2D 15N-HSQC stanexp/12 hnhagp3d; 3D HNHA (for 3 J H(N)Ha [Phi] coupling constant) stanexp/13 hnhbgp3d; 3D HNHB (only if sample if pretty damn good) stanexp/14 noesyhsqcetf3gp3d; 3D 15N-separated NOESY stanexp/15 dipsihsqcgpsi3d; 15N-seperated TOCSY stanexp/16 tocsynhsqcwg3.go; 15N-seperated TOCSY from Gottfried (clean CITY) stanexp/17 hsqct1etf3gpsi.2; 15N-relaxation measurement (T1) stanexp/18 hsqct2etf3gpsi; 15N-relaxation measurement (T2) stanexp/19 hsqcnoef3gpsi; 15N-relaxation measurement (heter. NOE) stanexp/20 ji_1h-t1hni; 15N-relaxation measurement (T1) from Junji Iwahara for PRE measurements stanexp/21 ji_1h-t2hni; 15N-relaxation measurement (T2) from Junji Iwahara for PRE measurements stanexp/22 troesyetf3gpsi; 2D 15N-TROSY-HSQC for large molecules, preferably deuterated stanexp/23 ipap-hsqc2.jm; 15N-HSQC IPAP version for RDC measurements (1H-coupled in F1(N)) stanexp/24 troesyetf3gpsi; 2D 15N-TROSY-HSQC for large molecules, preferably deuterated stanexp/24 troesyetf3gpsi; 2D 15N-TROSY-HSQC for large molecules, preferably deuterated

5 15N/13C-labeled samples (in H 2 O): stanexp/31 hsqcetf3gpsi; 2D 15N-HSQC (15N/13C-labeled sample) with adiabatic 13C decoupling stanexp/32 hsqcetgpsi; 2D 13C-HSQC (non constant time) stanexp/33 hsqcctetgpsisp; 2D 13C-CT-HSQC (constant time) stanexp/34 hnhagp3d; 3D HNHA stanexp/35 hnhbgp3d; 3D HNHB (only if sample if good) stanexp/36 noesyhsqcetf3gp3d; 3D 15N-separated NOESY stanexp/37 dipsihsqcgpsi3d; 15N-seperated TOCSY stanexp/38 tocsynhsqcwg3.go; 15N-seperated TOCSY from Gottfried (clean CITY) stanexp/39 Chsqcnoesy3.go; 3D 13C-separated NOESY (better in D 2 O, see below) stanexp/41 hncagpwg3d; 3D HNCA stanexp/42 hncagpwg3d check (const time version); 3D HNCA stanexp/43 hncocagpwg3d; 3D HN(CO)CA stanexp/44 hncacbgpwg3d; 3D HNCACB stanexp/45 cbcaconhwg3d; 3D CBCA(CO)NH stanexp/46 ccconhgp3d; 3D CC(CO)NH -TOCSY stanexp/47 hncogpwg3d; 3D HNCO stanexp/48 hncacogpwg3d; 3D HN(CA)CO stanexp/49 hccconhgpwg3d2; 3D HCC(CO)NH TOCSY stanexp/50 hbhaconhgpwg3d.t1.be; 3D HBHA(CO)NH stanexp/51 ipap-hnco2.jm; HNCO IPAP version for RDC measurements (1H-coupled in F1(C )) stanexp/52 ipap-hsqc2.jm; 15N-HSQC IPAP version for RDC measurements (1H-coupled in F1(N)) 15N/13C-labeled samples (in D 2 O): stanexp/61 Chsqcnoesy3.go; 3D 13C-separated NOESY stanexp/62 hcchdigp3d.2; 3D HCCH-TOCSY stanexp/63 hcchcogp3d; 3D HCCH-COSY stanexp/64 hacahbcosygp3d; 3D HACAHB-COSY (for 3 J HaHb [Chi1] coupling constants) stanexp/65 hbcbcgcdhdgp; 3D HBCBCGCDHD (for aromatic assignment) stanexp/66 hbcbcgcdcehegp; 3D HBCBCGCDCEHE (for aromatic assignment) stanexp/67 hcangp3d; 3D HCAN (can make sequential connections even through Pro) stanexp/68 hcacongp3d; 3D HCA(CO)N (can make sequential connections even through Pro) stanexp/69 hsqcctetgpjclr; 2D 13C-CT-HSQC (for 3 J C'Cb [Phi] coupling constants) stanexp/70 hsqcetfpf3gpjcsi; 2D 13C-CT-HSQC (for 3 J NCg [Chi1] coupling constants)

6 10%13C-labeled samples (in H 2 O): stanexp/71 hsqcctetgpsisp; 2D 13C-CT-HSQC (for Leu/Val methyl group stereospecific assignments; constant time using T = 26.6 ms; doublets will have negative intensity) Protein complexes (with one component 15N/13C-labeled and the partner unlabeled): stanexp/101 noesyf3gpphxf19.jm; 2D double half filtered NOESY (13C/15N) stanexp/102 noesyf3gpphxf19.jm; 2D double half filtered NOESY (13C/13C) stanexp/103 noesygpphwgxf.2; 2D 15N/13C filtered (F1, F2) NOESY stanexp/104 noesygpphxf19; 2D double half filtered NOESY stanexp/105 noesygpphxf19.jm; 2D double half filtered NOESY with 15N decoupling in F2 stanexp/106 hsqcgpnowgx33d; 3D 15N/13C -filtered (F3) 13C-separated (F1) NOESY stanexp/107 noesyhsqcgpwgx13d.ber; 3D 15N/13C -filtered (F1) 13C-separated (F2) NOESY stanexp/108 dipsi2gpphwgxf; 2D 15N/13C filtered (F1, F2) TOCSY