Automated backbone NMR resonance assignment of large proteins using redundant linking from a single simultaneous acquisition.
Related Articles Automated backbone NMR resonance assignment of large proteins using redundant linking from a single simultaneous acquisition.
J Am Chem Soc. 2020 Mar 04;:
Authors: Stanek J, Schubeis T, Paluch P, Güntert P, Andreas LB, Pintacuda G
Abstract
Thanks to magic-angle spinning (MAS) probes with frequencies*of 60-100 kHz, the benefit of high sensitivity*1H detection can now*be broadly realized in biomolecular solid-state NMR for the analysis of*microcrystalline,*sedimented, or lipid-embedded preparations. Nonetheless, performing*the assignment of all resonances remains*a*rate-limiting step in protein*structural studies, and even the latest optimized protocols fail to perform this*step when the protein size exceeds ~20 kDa. Here we leverage the benefits of*fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that*lifts this limitation. Through the creation, conservation and*acquisition of*independent magnetization pathways within a single triple-resonance MAS NMR*experiment, a single self-consistent dataset can be acquired, providing enhanced sensitivity, reduced vulnerability to*machine or sample instabilities,*and highly redundant linking that supports fully-automated peak picking and resonance assignment. The method, dubbed RAVASSA (Redundant Assignment Via A Single*Simultaneous Acquisition), is demonstrated with the assignment of the largest protein*to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated*microcrystalline sample and one week of spectrometer time.
PMID: 32129995 [PubMed - as supplied by publisher]
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