Bioinformatics Advance Access originally published online on September 11, 2006
Bioinformatics 2006 22(22):2833-2834; doi:10.1093/bioinformatics/btl477
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SCAssign: a sparky extension for the NMR resonance assignment of aliphatic side-chains of uniformly 13C,15N-labeled large proteins
1 Graduate Program in Bioengineering, National University of Singapore 10 Medical Drive, 117597 Singapore
2 Department of Biological Sciences, National University of Singapore 14 Science Drive 4, 117543 Singapore
*To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT 1 INTRODUCTION2 CORE FEATURES AND...3 RESULTS AND CONCLUSIONREFERENCES |
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Summary: SCAssign (side-chain assignment) is a Sparky extension written in Python to assist the NMR resonance assignment of aliphatic side-chains of uniformly 13C,15N-labled large proteins. It is based on a general strategy recently developed in our laboratory that makes use of 4D 13C,15N-edited NOESY, 3D MQ-(H)CCmHm-TOCSY, and prior backbone assignments. The program runs on all operating systems for which Sparky is available, and is easy to install, setup and use. Not only can it accelerate the assignment process, it also allows assignments of weak NOEs in 4D NOESY, which used to be very difficult with manual approach.
Availability: The program, in the form of source code, is provided as free download at http://yangdw.science.nus.edu.sg/SCAssign. The website also contains installation guide, user manual and demonstrations recorded in Flash.
Contact: Bug reports, questions or comments should be sent to dbsydw{at}nus.edu.sg.
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1 INTRODUCTION |
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TOPABSTRACT1 INTRODUCTION2 CORE FEATURES AND...3 RESULTS AND CONCLUSIONREFERENCES |
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Resonance assignment is often the most challenging step in NMR study of protein 3D structure. For proteins below 25 kDa, backbone and side-chain resonances can be assigned using uniformly 13C,15N-labeled samples and triple resonance experiments (Bax, 1994). Assignment of backbone and 13Cß resonances in larger proteins has been achieved with deuteration and TROSY techniques (Pervushin et al., 1997; Yang and Kay, 1999). Unfortunately, deuteration also severely reduces the number of NOE-derived distance constraints. Although, protein global folds can be calculated from only backbone HN–HN NOEs and residual dipolar couplings (Giesen et al., 2003), more accurate structure determination has to rely on NOEs among side-chain protons. The assignment of side-chain resonances in protonated large proteins, therefore, becomes absolutely necessary.
Our group has recently proposed a general strategy to assign aliphatic side-chain resonances of all residues in uniformly 13C,15N-labeled large proteins (Xu et al., 2005). It makes full use of 4D 13C,15N-edited NOESY, 3D MQ-(H)CCmHm-TOCSY (Yang et al., 2004), and prior backbone and 13Cß assignments. Most aliphatic side-chain resonances can be reliably assigned in this way. The manual process, however, is tedious and time-consuming. Here, we introduce a software tool designed to facilitate the SCAssign. Written in the Python language as a Sparky extension (T. D. Goddard, and D. G. Kneller, SPARKY 3, University of California, San Francisco), SCAssign runs on virtually all operating systems for which Sparky is available, and is easy to install, setup and use.
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2 CORE FEATURES AND IMPLEMENTATION |
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TOPABSTRACT1 INTRODUCTION2 CORE FEATURES AND...3 RESULTS AND CONCLUSIONREFERENCES |
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SCAssign provides a simple and user-friendly interface. Frequent tasks can be completed in main window (Fig. 1A) with minimum mouse-clicks. As screen space is precious during NMR spectral analysis, separate pop-up windows are used for setting spectra and preferences (Fig. 1B) and importing prior backbone assignments (Fig. 1C). SCAssign is able to correct the imported 13C chemical shifts for deuterium isotope effect (Venters et al., 1996). Some core features of the program are described below.
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2.1 Peak match
In principle, to assign the kth proton of residue i, SCAssign searches for NOE peaks whose chemical shifts match the shifts [
(HNi), (Ni), (
)] or [(HNi+1), (Ni+1), ()]. The user can specify such (HN, N, C) spin triplets using the pull-down menus in SCAssign's main window (Fig. 1A). Chemical shifts of HN, N, C
, and Cß are taken from prior backbone assignments. Since exact shifts of C
, C
and C
are unknown, their empirical ranges are used for locating possible peaks (The program includes chemical shift statistics of 20 amino acids from the BMRB restricted set). All matching peaks are sorted by data height and listed in SCAssign's peak list (Fig. 1A). The program will also fill in empirical ranges for the prior unassigned C and Cß and label them red in the pull-down menus, so that the user can try to assign them after most side-chain resonances have been assigned.
2.2 Dual view of spectrum
Suppose a few peaks are found to match [(HNi), (Ni) and (Ci)] due to spin triplet degeneracy. When the user clicks on any candidate in the peak list, besides showing the peaks on a C–H plane defined by spin pair Ni/Hi, the program will automatically display, in a separate view, another C–H plane located at Ni+1/Hi+1, and position the crosshair at [(Ci) and (Hi)] (Fig. 1D). Since an assignment obtained from an intraresidue NOE can be confirmed if it is consistent with that obtained from a sequential NOE, this will help the user quickly confirm assignments or resolve ambiguities.
2.3 Strip plot
Very often strip plots in CCH-TOCSY need to be combined with 4D NOESY to reliably assign C/H, C/H, and C/H spins. To show the CCH-TOCSY strip defined by an NOE peak, simply right click on its entry in the SCAssign peak list (Fig. 1A). The program calculates the aliphatic C/H frequencies of the peak, and passes them to the ‘Strip Plot’ extension (one of the standard extensions in Sparky package) for drawing the strip. Each strip is labeled with the C/H frequencies and the identity of the CH-containing residue (Fig. 1E). The position on the y-axis of the strip which corresponds to the 13C shift of a given NOE peak is marked for easy comparison of spectral patterns. The user is advised to plot the strips of C/H and Cß/Hß once they have been unambiguously assigned, so that later on when assigning C/H, C/H and C/H of the same residue, the user can plot the strip for each of the possible NOE peaks, and resolve the ambiguities by comparing the strips among themselves and with those of C/H and Cß/Hß, based on the matching of aliphatic 13C resonances.
2.4 Assignment and alias
Instead of manually filling in the residue and atom names, the user can assign a peak by Shift-clicking (press and hold the ‘Shift’ key while click) either on its entry in the peak list, or on its corresponding strip. When a peak is assigned, SCAssign will check its on-spectrum frequencies and, if necessary, auto-alias it using prior backbone assignments or empirical ranges as a guide. The user may edit the assignment label and aliases of a peak at any time via the usual Sparky commands.
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3 RESULTS AND CONCLUSION |
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TOPABSTRACT1 INTRODUCTION2 CORE FEATURES AND...3 RESULTS AND CONCLUSIONREFERENCES |
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We have tested SCAssign on a 42 kDa maltose binding protein and a 65 kDa chain-selectively labeled hemoglobin. The program worked stably and effectively under all conditions. SCAssign that used to take weeks can now be done within a day or two. In addition, it is doable to display the 4D NOESY spectrum at a much lower threshold as a result of automated peak matches and strip plots. This allows more side-chain resonances at
, and positions to be assigned from weak NOEs. Since many protons at the distal end of side-chains are also involved in medium- to long-range NOEs, their assignments can provide more high-quality distance constraints for accurate structure determination of large proteins. |
Acknowledgments |
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The authors thank Dr. Xu Yinqi and Mr. Zheng Yu for helpful discussions. This research was supported by a grant from the Biomedical Research Council (BMRC), the Agency for Science, Technology and Research, A*STAR of Singapore.
Conflict of Interest: none declared.
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FOOTNOTES |
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Associate Editor: Thomas Lengauer
Received on July 11, 2006; revised on September 4, 2006; accepted on September 4, 2006
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REFERENCES |
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TOPABSTRACT1 INTRODUCTION2 CORE FEATURES AND...3 RESULTS AND CONCLUSIONREFERENCES |
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Bax, A. (1994) Multidimensional nuclear magnetic resonance methods for protein studies. Curr. Opin. Struct. Biol, . 4, 738–744[CrossRef].
Giesen, A.W., et al. (2003) Determination of protein global folds using backbone residual dipolar coupling and long-range NOE restraints. J. Biomol. NMR, 25, 63–71[CrossRef][Web of Science][Medline].
Pervushin, K., et al. (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl Acad. Sci. USA, 94, 12366–12371
Venters, R.A., et al. (1996) Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II. J. Mol. Biol, . 264, 1101–1116[CrossRef][Web of Science][Medline].
Xu, Y., et al. (2005) A general strategy for the assignment of aliphatic side-chain resonances of uniformly 13C,15N-labeled large proteins. J. Am. Chem. Soc, . 127, 11920–11921[CrossRef][Web of Science][Medline].
Yang, D. and Kay, L.E. (1999) TROSY triple-resonance four-dimensional NMR spectroscopy of a 46 ns tumbling protein. J. Am. Chem. Soc, . 121, 2571–2575[CrossRef].
Yang, D., et al. (2004) Sequence-specific assignments of methyl groups in high-molecular weight proteins. J. Am. Chem. Soc, . 126, 3710–3711[CrossRef][Web of Science][Medline].
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