Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 113, Issue 33, Pages 9187-9192Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1602248113
Keywords
NMR spectroscopy; magic-angle spinning; protein structures; proton detection; viral nucleocapsids
Categories
Funding
- CNRS [IR-RMN FR3050]
- CNRS (Fondation pour la Chimie des Substances Naturelles)
- European Union [FP7-PEOPLE-2012-ITN 317127]
- European Research Council under the European Union's Horizon Research and Innovation Programme [648974]
- three Marie Curie incoming fellowships (Research Executive Agency Grant) [624918, 661175, 661799]
- European Molecular Biology Organization fellowship [ALTF 1506-2014, LTFCOFUND2013, GA-2013-609409]
- Marie Curie Actions (MSCA) [661175, 661799] Funding Source: Marie Curie Actions (MSCA)
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Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of H-1-H-1 proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.
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