Hi David,



I think this might be your fault:



"This is perhaps not surprising since I did not pick any peaks in the HNcoCACB, only in the HNCACB."



You have to pick the peaks in both spectra, so that Analysis can compare the chemical shifts on CA(i), CB(i) and CO(i) from the HNCACB and HNcaCO with CA(i-1), CB(i-1), and CO(i-1) from the HNcoCACB and HNCO.  I know you have picked both I and i-1 in the HNCACB, but I think Analysis might be set up to take i from HNCACB and i-1 from HNcoCACB ...



HTH



Markus



-----Original Message-----

From: CcpNmr software mailing list [mailto:[log in to unmask]] On Behalf Of David Thorn

Sent: Tuesday, September 11, 2012 3:44 AM

To: [log in to unmask]

Subject: sequential assignment



Dear all,



I am having problems setting up CCPN 2.2.2 for the sequential backbone assignment of a 278-residue protein. The project was set up along the guidelines suggested in the tutorial found here:



https://sites.google.com/site/ccpnwiki/Home/documentation/ccpnmr-analysis/tutorials/protein-assignment



I will summarise each step taken in the hope that someone could kindly point out where I went wrong... I started with HSQC, HNCACB, HNcoCACB, HNcaCO and HNCO spectra. The root peaks were initialised and then I proceeded to "Pick & Assign from Roots" the peaks in the HNCACB and HNcaCO. 4 peaks in the HNCACB and 2 peaks in the HNcaCO were linked to the same spin system. Additionally, the Ca, Cb and CO for each spin system were assigned as 'Ca', 'Cb' and 'C', respectively. The inter-residual peaks were picked but left unassigned. As such, for each spin system, I have up to 8 resonances, i.e. Ca, Cb, C, H, N, and another 3 corresponding to the inter-residual peaks which have no atom assignment. I then attempted to do the sequential assigment as described here:



https://sites.google.com/site/ccpnwiki/Home/documentation/ccpnmr-analysis/tutorials/protein-assignment/sequential-assignment



The first indication that something was wrong was that, in "M: Assignment: Protein Sequence Assignment", the HCN was not selectable in the 13C Window (only the 1H Window). However, NCH is selectable. Now the tutorial suggests to query HNcoCACB and match with HNCACB, but the resulting Spin System Table is empty. This is perhaps not surprising since I did not pick any peaks in the HNcoCACB, only in the HNCACB. The program will not allow me to both query and match the HNCACB.



With consideration for how I have picked and assigned peaks in the HNCACB and HNcaCO, could anyone please suggest how to set up the project so I can run the sequential assignment?



Many thanks,

David