SAGA: rapid automatic mainchain NMR assignment for large proteins

Abstract

Here we describe a new algorithm for automatically determining the mainchain sequential assignment of NMR spectra for proteins. Using only the customary triple resonance experiments, assignments can be quickly found for not only small proteins having rather complete data, but also for large proteins, even when only half the residues can be assigned. The result of the calculation is not the single best assignment according to some criterion, but rather a large number of satisfactory assignments that are summarized in such a way as to help the user identify portions of the sequence that are assigned with confidence, vs. other portions where the assignment has some correlated alternatives. Thus very imperfect initial data can be used to suggest future experiments.

Chemical shift assignments of domain 4 from the phosphohexomutase from Pseudomonas aeruginosa suggest that freeing perturbs its coevolved domain interface

“…Assignment of backbone amide, Cα, and Cβ resonances relied on a suite of triple resonance spectra expedited by the BEST approach: HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB, and HN(CO)CACB (Lescop et al 2007). Backbone resonances were interpreted with CARA (Keller 2004), SAGA (Crippen et al 2010), and Sparky (Goddard and Kneller 2000). Ambiguities remaining in sequential connections were resolved using (H)N(CAN)NH (Weisemann et al 1993).…”

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

“…In conclusion, considering the aforementioned unique assets, the use of APSY-NMR experiments in combination with a suitable automated assignment routine, as exemplified here with UNIO-MATCH-2014, is a valid alternative to the many previously proposed approaches for automated polypeptide backbone NMR assignment in proteins (e.g., Atreya et al 2000; Bartels et al 1997; Crippen et al 2010; Fredriksson et al 2012; Lee et al 2014; Lemak et al 2008; Lescop and Brutscher 2009; Moseley et al 2001; Schmidt and Güntert 2012; 2013; Schmucki et al 2009; Staykova et al 2008; Tikole et al 2012; Zawadzka-Kazimierczuk et al 2012; Zimmermann et al 1997). 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.…”

Apoprotein Structure and Metal Binding Characterization of a de Novo Designed Peptide, α3DIV, that Sequesters Toxic Heavy Metals

“…Backbone assignments were determined from a series of complementary three-dimensional (3D) triple resonance transverse relaxation optimized spectroscopy (TROSY) 27–29 experiments: HNCO/HN(CA)CO, 30 HN-(CO)CA/HNCA, 31 HN(CO)CACB/HNCACB, 32,33 and HN-(CA)HA 34 Since the sequence of α 3 DIV is known, the backbone assignments were manually completed and then successively confirmed with an autoassignment program SAGA 35 Ser24, Thr16, and Ser40 were used as landmarks in the manual assignment because the β -carbons of Ser and Thr residues are downfield of their α -carbons. Next, aliphatic assignments were obtained from the combined inphase/antiphase spectra of 3D HC(C)H-correlation spectroscopy (COSY) 36 and (H)CCH-total correlation spectroscopy (TOCSY) 37,38 experiments.…”

“…Next, aliphatic assignments were obtained from the combined inphase/antiphase spectra of 3D HC(C)H-correlation spectroscopy (COSY) 36 and (H)CCH-total correlation spectroscopy (TOCSY) 37,38 experiments. These assignments were further confirmed with 3D ( 1 H, 15 N, 1 H) nuclear Overhauser effect spectroscopy (NOESY)-TROSY 39 and ( 13 C, 15 N, 1 H) hetero-nuclear multiple-quantum coherence (HMQC)-TROSY experiments, providing a through-space validation of the COSY and TOCSY assignments.…”