1. NMR Spectroscopy Question and Answer Session
Judith Klein-Seetharaman Department of Structural Biology

2. What did we do? NMR instrumentation
NMR sample preparation considerations
Water signals
Water suppression
Detergents
NMR setup – downstairs zg
zgpr
selabs
hsqc
Spectral processing: Topspin Software
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

3. What did we do then?
Converting raw spectral formats to other formats using NMRpipe
Example: HIV protease relaxation data
Assignment of signals with one strategy
HNCO
HNCOCACBG
HNCOCA
HNCACB
HNCA
HNCACO
Example: Ubiquitin assignment with NMRviewJ
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

4. What would be the next step?
For determining a structure with NMR spectroscopy, after assignment, we would…
Collect constraints:
NOE
Dipolar coupling
Scalar coupling constants (gives dihedral angles)
Solvent exchange
Calculate a structure based on these constraints
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

5. What are other things one can do with NMR?
For study of dynamics
Measure and analyze T1, T2, HetNOE data
We went through T1 analysis in computer lab (conversion of raw data with NMRpipe, analysis with NMRviewJ)
T2 was your homework
Other studies might include chemical shift perturbations
Temperature titrations …
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

6. Homework Assignments 12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

7. Homework Part 1 NMR instrumentation
NMR sample preparation considerations
Water signals
Water suppression
Detergents
NMR setup – downstairs zg
zgpr
selabs
hsqc
Spectral processing: Topspin Software
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

8. Experiments Run zg = records all 1H's in your sample
zgpr = same as zg but with water suppression
selabs = the way we set it up, it records only NH region
hsqcfpgpf3gphwg = in short: hsqc = records all 1H's attached to 15N
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

9. click on Topspin icon and open the program
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

10. click on guest, expand MB3_Jan2007 directory
This is the directory that contains all of the data that was acquired in class.
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

11. Right click on MB3_Jan2007 12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

12. Select Expand & Show PULPROG/Title
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

13. Samples Sample IDs Experiment IDs B 1-7 F 10-15 A 20-23 D 30-33H
B 60, 61
F 70, 71 F B
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

14. Samples 40 = zg of sample E 41 = zgpr of sample E 1 = zgpr of Sample B
42 = selabs of Sample E
43 = hsqc of Sample E
50 = zg of Sample H
51 = zgpr of Sample H
52 = selabs of sample H
53 = hsqc of sample H
60 = zg of sample B
61 = zgpr of sample B
70 = zg of sample F
71 = zgpr of sample F
experiments done with comp bio students
100 = zg of sample F
101 = zgpr of sample F
102 = selabs of sample F
103 = hsqc of sample F
110 = zg of sample B
111 = zg of sample B
112 = selabs of sample B
113 = hsqc of sample B
Samples
1 = zgpr of Sample B
2 = zgpr of Sample B
3 = hsqc of Sample B
4 = zg of Sample B
5 = zg of Sample B
6 = selabs of Sample B
7 = selabs of Sample B
10 = zg of Sample F
11 = selabs of Sample F
12 = zgpr of Sample F
13 = hsqc of sample F
14 = hsqc of sample F
15 = hsqc of sample F
----- experiments done with MB3 students:
20 = zgpr of Sample A
21 = zg of sample A
22 = selabs of sample A
23 = hsqc of sample A
30 = zg of Sample D
31 = zgpr of Sample D
32 = selabs of Sample D
33 = hsqc of Sample D
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

15. Solving the Mystery of the Samples
Drag the experiment that you want to analyze into the main purple window area.
If you do not see a spectrum, you need to type efp
This will process the data so that you can see the spectrum.
In the case of the hsqc it will also ask you for an experiment number where to store the processed data, you can use the default or enter a number,
e.g. 2. Another alternative is to type “rser 1” that will retrieve the first slice of the 2d hsqc experiment also.
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

16. To see what acquisition parameters were chosen, click on the AcquPars tab, you will see a window:
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

17. Notice that you can scroll down to see many fields under this tab.
If you type ased only those parameters relevant to the specific experiment are shown:
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

18. Some tips
If you want to move more quickly from experiment to experiment, type re and then the experiment number, e.g.
re 21 gives the experiment stored in directory 21
To save a picture of a spectrum select File, Export and then give it a file name extension indicating which format you would like (e.g. .jpg)
Note that you may have to phase your spectrum.
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

19. Phasing
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

20. Sample A
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

21. 12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

22. Overlay Sample A
From these spectra, we can conclude that we do not have 15N label, it i a water sample. It does not have detergent in it, or the detergent is deuterated.
22 = selabs
21 = zg
20 = zgpr
23 = hsqc
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

23. Sample B. 1-7, 60-61, 110-113 3 = hsqc 8 = selabs 4 = zg 2 = zgpr
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

24. Sample D. 30-33 33 = hsqc 32 = selabs 31 = zgpr 30 = zg 12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

25. Sample E. 40-43 42 = selabs 41 = zgpr 40 = zg 43 = hsqc 12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

26. Sample F. 10-15, 70-71, 100-103 103 = hsqc 102 = selabs 101 = zgpr
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

27. Sample H. 50-53 52 = selabs – disregard this spectrum 53 = hsqc
51 = zgpr
50 = zg
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

28. Parameters to watch out forns rg
will affect actual intensity measured.
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

29. Several different assignment strategies exist
Most easily automated:
HNCO
HNCOCACB
HNCOCA
HNCACB
HNCA
HNCACO
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

30. Homework Part 2
Converting raw spectral formats to other formats using NMRpipe
Example: HIV protease relaxation data
Assignment of signals with one strategy
HNCO
HNCOCACBG
HNCOCA
HNCACB
HNCA
HNCACO
Example: Ubiquitin assignment with NMRviewJ
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

31. This is a cluster:
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

32. HNCO
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

33. HNCA
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

34. HNCACO
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

35. HNCOCACB, similar: CBCA(CO)NH
Not in HNCOCACB experiment
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

36. HNCOCA
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

37. HNCACB
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

38. What are other things one can do with NMR?
For study of dynamics
Measure and analyze T1, T2, HetNOE data
We went through T1 analysis in computer lab (conversion of raw data with NMRpipe, analysis with NMRviewJ)
T2 was your homework
Other studies might include chemical shift perturbations
Temperature titrations …
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

39. Dynamics in folded/unfolded lysozyme
Smaller rates – more flexible
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

40. T1 for HIV protease
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

41. T2 for HIV protease
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

42. T2 for HIV protease
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

43. From last class in case there are questions
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

44. NMR parameters Chemical Shift H2O methyl aromatic Trp-side-chain NH OH
Backbone NH
aliphatic
Side-chain HN Ha
Spectrum see handout
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

45. NMR of membrane proteins
In lipid bilayer:
In detergent micelle:
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

46. Problems! Detergent peaks
Detergent signals cause dynamic range problems
(Detergent signals cause spectral overlap)
Detergent deuteration is often not feasible
Problem: 1H,1H NOESY spectra do not show protein signals
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

47. Selective excitation 1H Chemical Shift [ppm] 1H Chemical Shift [ppm]
A. Selective excitation of the NH region using 90 degree pulse followed by direct observation.
B. Selective excitation of the same region as in A. Using excitation sculpting.
Backbone NH
Tryptophan side chain NH 20 15 10 5 10 5 -5
1H Chemical Shift [ppm]
1H Chemical Shift [ppm]
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

48. 2d HSQC
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

49. 1d projection of HSQC
12/9/2018
Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session