Highly automated protein backbone resonance assignment within a few hours: the "BATCH" strategy and software package.
Sequential resonance assignment represents an essential step towards the investigation of protein structure, dynamics, and interaction surfaces. Although the experimental sensitivity has significantly increased in recent years, with the availability of high field magnets and cryogenically cooled probes, resonance assignment, even of small globular proteins, still generally requires several days of data collection and analysis using standard protocols. Here we introduce the BATCH strategy for fast and highly automated backbone resonance assignment of (13)C, (15)N-labelled proteins. BATCH makes use of the fast data acquisition and analysis tools BEST, ASCOM, COBRA, and HADAMAC, recently developed in our laboratory. An improved Hadamard encoding scheme, presented here, further increases the performance of the HADAMAC experiment. A new software platform, interfaced to the NMRView software package, has been developed that enables highly automated NMR data processing and analysis, sequential resonance assignment, and (13)C chemical shift extraction. We demonstrate for four small globular proteins that sequential resonance assignment can be routinely obtained within a few hours, or less, in a highly automated and robust way.
Lescop
Ewen
E
Laboratoire de Chimie et Biologie Structurales, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, 1, Avenue de la Terrasse, 91190, Gif-sur-Yvette, France. ewen.lescop@icsn.cnrs-gif.fr
Brutscher
Bernhard
B
D000465:Algorithms; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012984:Software; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry; D014584:User-Computer Interface
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D025801:Ubiquitin