The J-UNIO protocol for automated protein structure determination by NMR in solution

Abstract

The J-UNIO (JCSG protocol using the software UNIO) procedure for automated protein structure determination by NMR in solution is introduced. In the present implementation, J-UNIO makes use of APSY-NMR spectroscopy, 3D heteronuclear-resolved [1H,1H]-NOESY experiments, and the software UNIO. Applications with proteins from the JCSG target list with sizes up to 150 residues showed that the procedure is highly robust and efficient. In all instances the correct polypeptide fold was obtained in the first round of automated data analysis and structure calculation. After interactive validation of the data obtained from the automated routine, the quality of the final structures was comparable to results from interactive structure determination. Special advantages are that the NMR data have been recorded with 6 to 10 days of instrument time per protein, that there is only a single step of chemical shift adjustments to relate the backbone signals in the APSY-NMR spectra with the corresponding backbone signals in the NOESY spectra, and that the NOE-based amino acid side chain chemical shift assignments are automatically focused on those residues that are heavily weighted in the structure calculation. The individual working steps of J-UNIO are illustrated with the structure determination of the protein YP_926445.1 from Shewanella amazonensis, and the results obtained with 17 JCSG targets are critically evaluated.

The Solution Structure and Dynamics of Full-length Human Cerebral Dopamine Neurotrophic Factor and Its Neuroprotective Role against α-Synuclein Oligomers

“…The NOE data were measured at 800 MHz or 1 GHz with a mixing time of 80 ms. The three NOESY spectra were peak picked and assigned automatically with the standard ATNOS/CANDID/CYANA protocol including seven cycles of peak picking, NOE assignment, and three-dimensional structure calculation with simulated annealing in torsion angle space (26). The final target function was 2.6 Å 2 .…”

Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N-Terminus of SOCS5

“…The software UNIO-ATNOS/CANDID 28,29 was used in combination with the torsion angle dynamics algorithm CYANA3.0 30 in a standard seven-cycle protocol for NOESY peak picking, NOE assignment and structure calculation. 31 The intensities from the NOEs were converted into distance restraints and combined with the backbone dihedral angles derived from TALOS, 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 17.5K. Dynamic exclusion was enabled for 90 s. …”

1H, 13C and 15N resonance assignments of a C-terminal domain of human CHD1

“…Out of the six assigned side chain amides, two isolated NH 2 groups of N64 and N74 are labelled. Regular secondary structure elements of CHD1C were identified from 13 C ab secondary shifts, which were calculated as previously described (Serrano et al 2012), and are reported in Fig. 2 Fig.…”