The 3D HNCACB experiment is specifically designed to correlate the ¹H and ¹⁵N amide resonances with those of the intra- and interresidue ¹³CA and ¹³CB resonances by means of the ¹J(NH), ^1,2J(N,CA) and optional ¹J(CA,CB) coupling constants. The same connectivities can be correlated with an analog 3D CBCANH experiment. In principle, the HNCACB experiment is more sensitive than CBCANH and is recommended for proteins with shorter T2 13C relaxation times and for deuterated proteins. However, the CBCANH experiment offers improved resolution in the 13C dimension because there is no J(CC) coupling evolution.

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 HNCO experiment must be recorded in H₂O.

The original 3D HNCACB pulse sequence ( 93JMRB201-101 ) consisted of the following steps:

1. Initial transfer from ¹H to ¹⁵N via ¹J(NH) using an INEPT pulse sequence.
2. Transfer from ¹⁵N to ¹³CA via ¹J(N,CA) using another INEPT pulse train.
3. A 90º ¹³C pulse transfers magnetization from ¹³CA to ¹³CB.
4. ¹³C chemical shift evolution during a variable t₁ period. followed by transfer magnetization from ¹³CB to ¹³CA.
5. Magnetization is transferred back to ¹⁵N
6. ¹⁵N chemical shift evolution during the constant-time t₂ period.
7. Finally, magnetization is transferred back to the NH protons by a retro-INEPT pulse train and proton acquisition is recorded under ¹⁵N decoupling.

Several improved versions have been proposed incorporating the following modifications:

• Improved sensitivity incorporating the PEP methodology in gradient-enhanced versions ( 94JMRB203-103 ).
• Improved sensitivity by ²H decoupling during the Constant-Time ¹³C evolution period  ( 94JACS11655 and 96JACS6570 ) in  ¹⁵N,¹³C,²H-labeled proteins.
• Improved sensitivity and resolution using the TROSY approach ( 99JACS844 , 01JB177-20 ).
• Sequential 3D HNCACB experiment to obtain only sequential connectivities ( 01JMR328-151 ), similar to the 3D HN(CO)CACB experiment but avoiding CO magnetization transfer.
• A 3D HNCACB-E.COSY pulse sequence to measure 3J(CB-CO) coupling constants.

The 3D HNCACB experiment can be recorded in automation mode. More details on practical implementation of the 3D HNCACB experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HNCACB experiment

The HNCACB experiment affords a 3D spectrum in which ¹H, ¹⁵N and ¹³CA/¹³CB chemical shifts are displayed in three independent dimensions. Two different connectivities are present:

• Intra-residue correlations due to ¹J(NH) +  ¹J(N,CA) + optional ¹J(CA,CB).
• Sequential correlations due to ¹J(NH) +  ²J(N,CA) + optional ¹J(CA,CB).

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