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<p>Created page with &#039; == &#039;&#039;&#039;Amide Side-chain Resonance Assignment&#039;&#039;&#039; == You will normally need to have 3 windows open at the same time: # Open aliphatic 2D [13C, 1H]-HSQC with &#039;&#039;&#039;Open PolyScope&#039;&#039;&#039;…&#039;</p> <p><b>New page</b></p><div><br /> <br /> == '''Amide Side-chain Resonance Assignment''' ==<br /> <br /> You will normally need to have 3 windows open at the same time:<br /> <br /> # Open aliphatic 2D [13C, 1H]-HSQC with '''Open PolyScope''' and select aliphatic 13C-resolved NOESY for the strip display. This is similar to displaying 3D HNCO or 3D 15N-resloved NOESY paired with a 2D [15N, 1H]-HSQC.<br /> # Open 3D 15N-resolved NOESY with '''Open SystemScope'''. Rotation is normally not needed, since the default dimension order is '''Hinept''' = '''X*, *Hnoe''' = '''Y*, *Ninept''' = *Z*. Thus, the left panel of SystemScope will contain an H-H plane (with Hnoe dimension vertical), and the right panel will contain the H-N plane.<br /> # Open 2D [15N, 1H]-HSQC with '''Open PolyScope''' and select 15N-resolved NOESY for the strip display. <br /> <br /> === Asn ===<br /> <br /> # Open an HB2/CB or HB3/CB strip in window 1.<br /> # Pick a HD21 candidate. This should be medium peak around 6 - 7 ppm. Make sure the peak is not from the backbone H of the next residue (though, overlap is possible).<br /> # In window 2 display the H/N strip of the same Asn residue.<br /> # Select the HD21 candidate. Right-click and choose '''Show Orthogonal''' in the pop-up menu.<br /> # In the orthogonal plane pick an ND2 candidate. The correct position should exhibit HD21-HB2 and HD21-HB3 intra NOE peaks.<br /> # In window 3 verify and adjust the position of HD21/ND2 spin pair.<br /> # In window 3 pick the HD22 spin. This is usually the strongest peak in the strip.<br /> # In window 3 verify and adjust the position of HD22/ND2 spin pair.<br /> <br /> Due to rigidity of the -CONH2 group HD21 and HD22 are practically never degenerate. Also, only one of them will display strong NOE peaks to HB2 and HB3.<br /> <br /> === Gln ===<br /> <br /> # Open an HG2/CG or HG3/CG strip in window 1.<br /> # Pick a HE21 candidate. This should be medium peak around 6 - 7 ppm. Make sure the peak is not from the backbone H of the next residue (though, overlap is possible).<br /> # In window 2 display the H/N strip of the same Gln residue.<br /> # Select the HE21 candidate. Right-click and choose '''Show Orthogonal''' in the pop-up menu.<br /> # In the orthogonal plane pick an NE2 candidate. The correct position should exhibit HE21-HG2 and HE21-HG3 intra NOE peaks.<br /> # In window 3 verify and adjust the position of HE21/NE2 spin pair.<br /> # In window 3 pick the HE22 spin. This is usually the strongest peak in the strip.<br /> # In window 3 verify and adjust the position of HE22/NE2 spin pair.<br /> <br /> Due to rigidity of the -CONH2 group HE21 and HE22 are practically never degenerate. Also, only one of them will display strong NOE peaks to HG2 and HG3.<br /> <br /> === Arg ===<br /> <br /> # Open an HD2/CD or HD3/CD strip in window 1.<br /> # Pick a HE candidate. This should be medium peak around 6 - 7 ppm. Make sure the peak is not from the backbone H of the next residue (though, overlap is possible).<br /> # In window 2 display the H/N strip of the same Arg residue.<br /> # Select the HE candidate. Right-click and choose '''Show Orthogonal''' in the pop-up menu.<br /> # In the orthogonal plane pick an NE candidate. The correct position should exhibit HE-HD2, HE-HD3, possibly HE-HG2 and HE-HG3 intra NOE peaks. Remember that NE is most likely folded, since 15N dimension is usually 25-35 ppm wide, centered around 115-120 ppm, while NE is found near 80-100 ppm.<br /> # In window 3 verify and adjust the position of HE/NE spin pair. The correct folding of NE can be checked in a 2D [15N, 1H] HSQC with a wide spectral width.<br /> <br /> HE spins may not be observed for all Arg residues. <br /> <br /> <br /> <br /> <br /> <br /> <br /> -- Main.AlexEletski - 01 Nov 2007</div>

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