Triple Resonance Backbone Assignment: HNCA

The HNCA experiment is useful for assigning the backbone when used with HN(CO)CA, HNCACB and CBCA(CO)NH.

In each NH strip, two peaks of unequal intensity can be observed. The more intense peak belongs to the same residue Ca (i) whereas the less intense peak (sometimes lost in the noise) belongs to the previous residue Ca (i-1). By connecting NH strips of i-1 to i residues we can perform the sequential walk.

With the help of HN(CO)CA, the i-1 residues can be easily identified by overlapping both HNCA and HN(CO)CA spectra.

Below is the overlaid spectra HN(CO)CA and HNCA of 15N, 13C-Ubiquitin sample recorded at 293K.