The 3D H(N)CA,CO-E.COSY experiment allows to measure several ³J(CO-X) coupling constants (X is the passive spin) in ¹³C,¹⁵N-labeled proteins in H₂O from E.COSY-like multiplet patterns.

Implementation on AVANCE spectrometers equipped with a third channel and pulsed field gradients (PFGs).

The original pulse sequence ( 97JB107 and 97JACS1468 ) consists of the following steps:

Initial transfer from NH to ¹⁵N using an INEPT block

Simultaneous evolution of the J(N-CA)and J(N-CO) couplings and transfer to ¹³CA using another INEPT sequence.

Evolution of ¹³CA chemical shift during a variable t₁ period followed by a second transfer to ¹³CO.

Evolution of ¹³CO chemical shift during a variable t₂ period and transfer back to ¹⁵N.

A ¹⁵N-to-NH retro-INEPT scheme incorporating a WATERGATE water suppresion block prior to NH detection under 15N decoupling.

Several variants have been proposed as a function of the mechanism involved during the t₁ and t₂ evolution periods in which the state of the corresponding passive spin has been preserved:

3D H(N)CA,CO[HA}-E.COSY experiment to measure J(CO-HA) coupling constants, in which HA is the passive spin ( 97JB107 , 97JACS1468 and 98JACS3803 ).

3D H(N)CA,CO[CB}-E.COSY experiment to measure J(CO-CB) coupling constants, in which CB is the passive spin ( 97JB107 ).

3D H(N)CA,CO[CO}-E.COSY experiment to measure J(CO-CO) coupling constants, in which CO is the passive spin ( 97JB107 ).

The 3D H(N)CA,CO-E.COSY experiment affords a 3D map with (F1,F2,F3=CA,CO,NH) in which coupling constants are extracted from E.COSY cross-peaks in the CA,CO plane