HNCACAB/CABCA(CO)NH

Backbone Assignment with XEASY/UBNMR

Sequential backbone and 13CB resonance assignment is associated with mapping of SRDs identified in spin system identification onto the polypeptide sequence. This is accomplished using two (4,3)D GFT NMR experiments, that is, HNNCABCA and CABCA(CO)NHN, or using two non-GFT experiments HNNCACB/CACBCONHN.

Analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra

The HNNCABCA contains peaks representing both intra-residue and sequential connectivities (as in HNNCACB). Since the sequential connectivities are often comparably weak, this experiment is routinely combined with CABCA(CO)NHN (which comprises, as CBCA(CO)NHN, sequential connectivities only).These can be used to sort SRDs in sequential order, and to then assign them to specific residues in the primary structure.

1. Creating and adjusting simulated peak lists for (4,3)D GFT spectra
   
2. Go to the analysis/xeasy/backbone directory and edit the macro getfil to import backbone spectra, SequenceList, AtomList and PeakLists.
3. Edit the makeCabcaPeak macro and update the sequence and atom list file names. In UBNMR, run the makeCabcaPeak script to generate an extended GFT AtomList and PeakList. The new AtomList contains linear combinations of 13CA and 13CB shifts for each residue and SRD. Intraresidue 13C shifts are assigned to SRD-I numbers, while 13C shifts of the residue preceding SRD-I are assigned to SRD-II numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process.
4. In XEASY, use ns, ls, lc, and lp to load HNNCABCA and CABCA(CO)NHN spectra and the SequenceList, AtomList and PeakList
   1. Use se, gs, fs and bs (as described here; you have to use se in each spectrum) to sort and display strips (Figure 1A)
   2. Use mr to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks)
   3. Use ra regularly to check on the quality of the PeakList; ac, wc and wp to save updated lists.

Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra
A: Before peak position adjustment by mr


B: After peak position adjustment by mr

1. Initial Backbone Assignment from AutoAssign
   
   1. Go to /analysis/xeasy/backbone/autos, read autos_README for instructions on how to edit the macro makeAutoList and file myprot.aat.
   2. Run makeAutoList in UBNMR to generate input files for AutoAssign. Errors at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running the makeAutoList script again, until all errors are eliminated. The peak ID the simulated AutoAssign input file (myprot-hsqc.pks) corresponds to the SRD-I number, which is required for this protocol.
      Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that you generate a 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results. However, please be aware that this protocol discards the 4D information which reduces the GFT (4,3)D spectra into the equivalent of conventional 3D HNNCACB and CBCA(CO)NHN.
      1. Peak pattern used in the AutoAssign input file for 3D CACB type experiments is: HN(i), N(i), CA(i or i-1) or CB(i or i-1);
      2. Peak pattern used in the AutoAssign input file for 4D CACB type experiments is: HN(i), N(i), CA(i), CA(i) or CB(i) and HN(i), N(i), CA(i-1), CA(i-1) or CB(i-1).
         
      3. Run AutoAssign several times with varying matching tolerances for CA and CB in the myprot.aat file (0.1-0.6ppm); save AutoAssign output files
         
      4. It is suggested to use Default Execution method to run the program.
      5. From the main menu of AutoAssign, select Examine > All GSs to write an output file that contains both AutoAssign assignment and the corresponding SRD-I residue numbers.
         
      6. In UBNMR, run macro AA2Xeasy to use the best or consensus AutoAssign output file to complement the SRD-I and SRD-II entries of the XEASY SequenceList with mapping numbers.
2. Confirming Backbone Assignment from AutoAssign in XEASY
   Here two XEASY sessions are recommended for efficiency, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
   1. In UBNMR, run macro makeBbNNoesy to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that only contains diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.

1. 1. In XEASY session I, ns to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use ls to load the SequenceList that contains AutoAssign results; lc to load the AtomList; use lp to load the CABCA-PeakList.
   2. In XEASY session II, use ns to load 15N-resolved NOESY; use ls to load the SequenceList that contains AutoAssign results; use lp to load the corresponding starting peak list. There is no need to move any peaks at this time.
   3. In XEASY session I and II, use sn to swap fragment number and mapping; use se to sort strips; use ls and lc to load the SequenceList (with AutoAssign assignment) and the atomlist; use gs to display the strips. Now the strips are sorted in the following order: Assigned SRD residues in sequential order followed by unassigned SRD residues in SRD -1 sequential order.
   4. In XEASY session I and II, check and confirm sequential connectivities for the assigned SRD residues. Use ed to modify the mapping number if the assignment is wrong.
   5. Continue to complete the backbone assignment manually as described below
2. Complete Backbone Assignment by perform sequential ordering of SRDs
   Here two XEASY sessions are recommended for efficiency reasons, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY.
   1. In XEASY session I, use es, se, gs to select two strips exhibiting four well resolved peaks arising from a Ser, Thr, Ala residue in (4,3)D HNNCABCA or terminal residues of an assigned segment assigned from AutoAssign as a starting point; use sh to put those strips "on hold".
   2. In XEASY session I, use rd and pc to search for sequential neigbours.
   3. In XEASY session I, using fc and bc to inspect the sorted strips in order to identify the strip containing the sequential neighbor and use sh to put "on hold".
   4. In XEASY session II, use cd / cc to confirm sequential connectivities in 15N-resolved NOESY.
   5. In XEASY, repeat steps 1 to 4 until you have identified a maximal set of strips you can map to the polypeptide sequence.
   6. In XEASY, use ed to edit peak entries, type in "mapping numbers" which link the SRD-I and SRD-II numbers to residue numbers.
   7. In XEASY, repeat 5 and 6 until analysis is complete
   8. In XEASY, use aa, ac, ws, wc and wp to save all XEASY files before switching from SRD to sequential amino acid residue numbering using sn. This yields modified SequenceList, AtomList and PeakList which should be saved, and re-loaded and saved a second time.
   9. In UBNMR, run cleanBbGftProt to make a clean AtomList and SeqList by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.

#UBNMR macro cleanBbGftProt

init

read seq nhsqc.seq

read prot bbgft-swapped.prot append

update atom GFTatom CA

update atom GFTatom CB

remove SRDs

write seq final-clean.seq

remove GFTatoms

write prot final-clean.prot

Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra

In case of NMR data were collected with (4,2)D GFT HNNCABCA and CABCA(CO)NHN for backbone assignment, one can treat them the same as previously described (4,3)D GFT HNNCABCA/CABCA(CO)NHN analysis. Since the protocol of analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra is based on analysis the stips residue by residue, it can be completely adapted to the analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra. Simply do the same things as described above:

1. Simulating the intial peak lists;
2. Adjusting peak position in strips of all residues with XEASY;
3. Calculating single quantum chemical shifts from GFT shift linear combinations
4. Simulating AutoAssign input files and do the initially backbone assignment by AutoAssign;
5. Maually checking results from AutoAssign and completing the assignment.