The 3D HN(CO)CA experiment is specifically designed to correlate ¹⁵N and NH chemical shifts with the interresidue ¹³CA carbon shifts via the intervening ¹³CO nucleus by means of the ¹J(NH), ¹J(N,CO) and ¹J(CA,CO) coupling constants. Intra- and interresidue connectivites can also be extracted from 3D HNCA experiments.

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)CA experiment must be recorded in H₂O.

The original 3D HN(CO)CA pulse sequence ( 91JB99 ) consisted of the following steps:

1. Initial transfer from ¹H to ¹⁵N via ¹J(NH) using an INEPT pulse sequence.
2. ¹⁵N chemical shift evolution during the variable evolution t₁ period.
3. Fixed evolution delay to achieve antiphase ¹⁵N magnetization with respect to ¹³CO via ¹J(N,CO) followed by a magnetization transfer to ¹³CO by simultaneous 90º ¹⁵N and ¹³CO pulses.
4. Fixed evolution delay to achieve antiphase ¹³CO magnetization with respect to ¹³CA via ¹J(CA,CO).
5. ¹³CA chemical shift evolution during the variable evolution t₂ period in an HMQC-type way.
6. Magnetization is transferred back to the NH protons by reversing the transfer steps described in points 4, 3 and 1.
7. Proton acquisition under ¹⁵N decoupling.

Several improved versions have been proposed incorporating the following modifications:

• Constant-time period (CT) in the F₁(¹⁵N) dimension, HSQC-like transfer in F₂(¹³CA) dimension and optional ¹H decoupling instead of 180º ¹H pulses ( 92JMR432-96 ).
• Incorporation of PFGs ( 92JMR638-99 ).
• Improved sensitivity incorporating the PEP methodology in phase-cycled ( 92JMR431-100 ) and gradient-enhanced versions ( 94JACS11655 ) similarly as described for the HNCO experiment ( 93ANG1489 , 94JMRB203-103 , and 94JMRA129-109 ).
• The variable evolution period in which ¹⁵N chemical shifts take place (see step 2) is placed after the variable evolution period of ¹³CA chemical shifts ( 94JACS11655 and 96JMRB91-113 ) as described for the HNCO experiment ( 93ANG1489 , 94JMRB203-103 , and 94JMRA129-109 ).
  
• Improved sensitivity by ²H decoupling during the Constant-Time CA evolution period in  ¹⁵N,¹³C,²H-labeled proteins ( 94JACS11655 ). The use of selective ¹³CB decoupling during this period has also been proposed ( 96JMRB91-113 ).
• Improved sensitivity and resolution using the TROSY approach ( 99JACS844 ).
• In overlapped spectra it could be advisable to record a slightly modified 3D H(N)COCA experiment:

• Other related experiments are:
  3D (HACA)CO,NH ( 96JMRB65-110 )
  2D HN(CO)(CA) ( 91JMR413-94 )
  3D HN[COCA] ( 95JB202 and 95JMRB197-108 )
  3D (HA)CA(CO)NH ( 97JB105 ) and 3D (H)CA(CO-TOCSY)NH ( 00JB127 ) experiments.
  2D HN(alpha/beta-COCA-J) experiment (99JMR44-141) to measure 1J(COCA) coupling constants

The 3D HN(CO)CA experiment can be recorded in automation mode. More details on practical implementation of the 3D HN(CO)CA experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HN(CO)CA experiment

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

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