Abstract
Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.
Highlights
Atomic-resolution structure determination is crucial for understanding protein function
Our structure features a long loop region in the catalytic chamber, comprising residues 119–138, shown in green in Fig. 4c, d. This loop has not been modeled in the crystal structure. While this loop region appears relatively well defined at cryo electron microscopy (cryo-EM) conditions, at lower local resolution, only methyl resonances of Val-120 and Ile-124 were detected by nuclear magnetic resonance spectroscopy (NMR) but we were unable to assign the backbone resonances of this loop at room temperature by Magic-angle spinning (MAS) NMR (Supplementary Fig. 2E; the short helix we modeled was inferred from the TALOS-N algorithm.) These findings may suggest that the loop is dynamically disordered at room temperature, leading to low signal intensity in MAS NMR experiments, and to non interpretable density in crystal structures determined at cryogenic a EM: helical density features α-helix-to-density assignments
8 Å EM map Discussion The recent “resolution revolution” in cryo-EM is progressively changing the way how the structures of biomolecular assemblies are determined, but currently de novo structure determination remains reserved to a minority of EM data sets
Summary
Atomic-resolution structure determination is crucial for understanding protein function. The situation is significantly more complex: the former approach, i.e., determining a rough fold from NMR and refining with EM data, is challenging because lack of NMR restraints—often the case for large proteins—precludes obtaining a converged structural model which could be used for EM-based refinement. The latter method suffers from the fact that de novo building of the protein sequence into an EM map, i.e., unambiguously identifying the residues and being able to follow the chain throughout the map, becomes increasingly complex with protein size, and very strongly depends on the resolution of the EM data
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