Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

Abstract

Atomic-resolution structure determination is the key requirement 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 enabled determination of the high-resolution structure 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 specifically labelled 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 (up to 8 Å) cryo-EM data are available, thus paving avenues for the structure determination of challenging biological assemblies.

Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs

“…Therefore we decided to use the automated assignment algorithm FLYA (Schmidt and Güntert 2012), which is part of the program CYANA (Güntert 2004). FLYA can easily incorporate all of the above-mentioned data into an efficient and automated resonance assignment procedure, even for large systems with complex input data (Gauto et al 2019). In addition, we analyzed the performance of the FLYA assignment procedure when supplying different subsets of the input data.…”

Asymmetric Synthesis of Methyl Specifically Labelled L‐Threonine and Application to the NMR Studies of High Molecular Weight Proteins

“…Full relaxation‐matrix analysis is required to extract accurate distance restraints from such 3D NOESY spectra . Such long‐range distance restraints are particularly useful for the structural investigation of large proteins …”

Comparison of the structure and function of a chimeric peptide modified titanium surface

“…Therefore, the antimicrobial effect of chimeric peptide is not directly proportional to its length and number of structural groups, and there is a certain relationship between its function and the number of functional groups in microenvironment. 39,40 XPS analysis of the binding elements' compositions of the Ti surface conrmed the presence of C 1s, O 1s (531.18 eV), Na 1s (1071.83 eV), P 2p (133.19 eV), C l2p (198.69 eV), and Ti 2p 3 (458.30 eV) as the chief components, as shown in Fig. 3.…”