We recently included solution NMR spectrometer as one of our main gear to study protein structure, function, and regulation. We now use solution NMR spectrometer to study small protein (40-150 residues) to understand the key structural information in terms of molecular recognition and dynamics. The Academia Sinica High Field NMR Center (AS-HFNMRC) houses 6 advanced spectrometers and we often access the NEO850 (850 MHz), AVANCEIII-800, NEO600, and AVANCEIII-600 for our daily experiments.

We also applied the hmsIST‘s non-uniform sampling scheduler to accelerate our data collection. Here are the suggest settings for the protein NMR experiments.

Expt	Topspin “par”	NUS sparsity	Notes
15N-HSQC	std1_2D_15N-HSQC _hsqcetfpf3gpsi	not applied
13C-HSQC	std1_2D_13C-HSQC _hsqcctetgpsp	not applied	d23=13.3ms (constant time) for opposite signs of CH3/CH vs CH2
13C-HSQC	std1_2D_13C-HSQC _hsqcetgpsi	not applied
HNCO	std1_3D_HNCO _hncogpwg3d	10%	backbone assignment
HNcaCO	std1_3D_HN(CA)CO _hncacogpwg3d	10%	backbone assignment
HNCA	std1_3D_HNCA _hncagowg3d	10%	backbone assignment
HNcoCA	std1_3D_HN(CO)CA _hncocagpwg3d	10%	backbone assignment
CBCAcoNH	std1_3D_CBCA(CO)NH _cbcaconhgpwg3d	10%	backbone assignment
HNCACB	std1_3D_HNCACB _hncacbgpwg3d	10%	backbone assignment
HBHAcoNH	std1_3D_HBHA(CO)NH _hbhaconhgpwg3d	10%	sidechain HB and HA for (i-1) residue F1 dimension shifted by (SW1)/4, i.e. SW1=8ppm, F1 is shifted by 2ppm.
HccoNH	std1_3D_HCCCONH-H _hccconhgp3d2	10%	sidechain protons for (i-1) residue. F1 dimension shifted by (SW1)/4, i.e. SW1=8ppm, F1 is shifted by 2ppm.
CcoNH	std1_3D_HCCCONH-C-hcccohgpwg3d3	10%	sidechain carbons for (i-1) residue
HCCH-TOCSY	std1_3D_HCCHTOCSY _hcchdigp3d	10%	sidechain protons. F1 dimension shifted by (SW1)/4, i.e. SW1=8ppm, F1 is shifted by 2ppm.
CCH-TOCSY	hcchdigp3d2	10%	sidechain carbons.
15N-TOCSY	std1_3D_15N_TOCSYHSQC-dipsihsqcf3gpsi3d	10%	sidechain protons for residue i, DISPI mixing time d9 = 60ms
HNcaNNH	HNCANNHGPWG3D	10%	default d27=12.5ms, it will show N(i+i) and N(i-1)
HNcaNNH.kpw		10%	d27=8ms (manually changed), it will show N(i), N(i+1), and N(i-1), where N(i) is opposite phase to the other two. Use KP’s modified pulse sequence
15N-NOESY	std1_3D_15N-NOESYHQC _noesyhsqcfpf3gpsi3d	25-35%	mixing time d8=120ms
13C-NOESY	std1_3D_13C-NOESYHSQC _noesyhsqcetgpsi3d	25-35%	mixing time d8=120ms

The above 3D NMR experiments often require 0.5-1.5 days for each. With the application of 10-35% NUS sparsity, we can reduce the data acquisition time from 360 hours to 34 hours for all NMR experiments listed above.

Here are some examples of the collected NUS-based spectra: