The 3D HN(CO)CACB experiment is specifically designed to correlate the ¹H and ¹⁵N amide resonances of one residue with both ¹³CA and ¹³CB resonances of its preceding residue via the intervening ¹³CO spin by means of the ¹J(NH), ¹J(N,CO), ¹J(CA,CO) and optional ¹J(CA,CB) coupling constants. A closely related experiment is the 3D CBCA(CO)NH experiment.

Implementation on AVANCE spectrometers equipped with a third channel. Improved versions using pulsed field gradients (PFGs) are also available and, therefore, in such cases gradient technology is required.

The experiment is applied on ¹⁵N,¹³C-labeled proteins. Because the amide (NH) protons are involved, the  HN(CO)CACB experiment must be recorded in H₂O.

The original 3D HN(CO)CACB pulse sequence ( 94JACS11655 ) consisted of the following steps:

Several improved versions have been proposed incorporating the following modifications:

• Use of 2H-decoupling duirng the evolution of CA and CB:
• Use of Watergate for improved solvent suppression in the last 15N-to-1H transfer step:
• Improved sensitivity and resolution using the TROSY approach with optional 2H decoupling:

More details on practical implementation of the 3D HN(CO)CACB experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HN(CO)CACB experiment

The HN(CO)CACB experiment affords a 3D spectrum in which ¹H, ¹⁵N and ¹³CA/¹³CB chemical shifts are displayed in three independent dimensions. Sequential connectivities are due to ¹J(NH) +  ¹J(N,CO) +  ¹J(CA,CO) + optional ¹J(CA,CB).

See list of 3D triple-resonance NMR experiments for doubly-labeled proteins.