CABM Standard Data Collection Procedure
for Protein Structure Determination


All spectra within a set should be run back-to-back on same spectrometer system.
Sets A1 and A2 should be run together, but definitely on same spectrometer system.
Sets B and C should be run together , but definitely on same spectrometer system.
Sets A1, A2, B, and C all at 600 MHz or greater field strength.


Set A1  Basic Backbone Assignmentswith AutoAssign
1. 2D [N,H] HSQC
2. 2D [N,H] HSQC -- large sw
3. 2D [N,H] HSQC – NH2 only
4. 2D [C,H] HSQC
5. 3D HNCACB
6. 3D HNCO
7. 3D HNcoCACB
8. 3D HNCA
9. 3D HNcoCA
10. HAcaNH -- Gly    2D NH plane and  3D version
11. HAcocaNH -- Gly   2D NH plane and 3D version

Set A2  Validation and Extenstion of Backbone Assignments
12. 3D hCccoNH-TOCSY  (tune for CH3, mix = 30 ms, nloop = 3)
13. ( 3D RD HCccoNH-TOCSY in place of 12 )
14. 3D HNcaCO (usually run as hacaCOcaNH)
15. 3D 15N-NOESY (also analyze as part of Set C)

Set B  Automated Sidechain Assignments
16. 3D hCCH-COSY
17. 3D HcCH-COSY
        (or 3D RD  HCCH-COSY in place of 16/17 )
18. 3D HcccoNH-TOCSY – different  mixing times
19. 3D hCccoNH-TOCSY -- different  mixing times (if needed based on 12)
        (3D or RD HCccoNH-TOCSY - different  mixing times, in place of 18/19)
20. 2D RD HBCBcgcdHD for aromatics
21. 2D RD 1H-TOCSY-relayed HCH-COSY
22. 2D TOCSY-[NH]-HSQC – with 15N-H suppression, H-H plane, look at aromatics

Set C   NOESY and RDC Data for AutoStructure
23. 3D 13C-NOESY - CABM Pulse Sequence  (aliphatic 13C excitation)
24. 3D 13C-NOESY - Protein Pak Sequence (aliphatic + aromatic 13D excit)
        (or 3D 13C-NOESY – CABM Sequence, aromatic 13C excitation)

25. 4D 13C-NOESY
26. RDC experiments

Set D   Using 10% 13C, 100% 15N Sample, Generally at 500 MHz
27. 2D HNOE (do early, while running Set A)
28. H/D Exchange with HSQC or Mexico?
29. 2D HSQC-J (4 – 5 points) or HNHA for 3J(HN-Ha) values
30. 2D [C,H] HSQC using 10% 13C sample for Methyl Stereoassignments
31. Other J and RDC experiments