J-UNIO protocol used for NMR structure determination of the 206-residue protein NP_346487.1 from Streptococcus pneumoniae TIGR4

Abstract

The NMR structure of the 206-residue protein NP_346487.1 was determined with the J-UNIO protocol, which includes extensive automation of the structure determination. With input from three APSY-NMR experiments, UNIO-MATCH automatically yielded 77% of the backbone assignments, which were interactively validated and extended to 97%. With an input of the near-complete backbone assignments and three 3D heteronuclear-resolved [1H,1H]-NOESY spectra, automated side chain assignment with UNIO-ATNOS/ASCAN resulted in 77% of the expected assignments, which was extended interactively to about 90%. Automated NOE assignment and structure calculation with UNIO-ATNOS/CANDID in combination with CYANA was used for the structure determination of this two-domain protein. The individual domains in the NMR structure coincide closely with the crystal structure, and the NMR studies further imply that the two domains undergo restricted hinge motions relative to each other in solution. NP_346487.1 is so far the largest polypeptide chain to which the J-UNIO structure determination protocol has successfully been applied.

APSY-NMR for protein backbone assignment in high-throughput structural biology

“…The Figure 3 illustrates that within the range covered by the 30 proteins of Table 1, the result of polypeptide backbone assignments based on the present protocol of using APSY-NMR and UNIO-MATCH-2014 does not depend critically on the protein size. Based on these results, it is not surprising, that the same approach has successfully been applied for proteins with about 200 amino acid residues (Jaudzems et al 2014; Mohanty et al 2014). …”

“…In spite of a court injunction which blocked the use of APSY and related techniques for several years (Wüthrich 2011), APSY-NMR has become a standard technique for projects of the Joint Center for Structural Genomics (JCSG: www.jcsg.org). Within the J-UNIO protocol for extensive automation of protein structure determination (Serrano et al 2012), APSY-NMR is routinely used for polypeptide backbone assignments of proteins with sizes up to about 150 amino acid residues, and significantly larger proteins have also been successfully studied (Jaudzems et al 2014; Mohanty et al 2014). Here we report the results obtained with a representative sample of 30 JCSG target proteins.…”