# Collecting Large Datasets of Unambiguous Structural Restraints for Protein Structure Determination by 4D Proton‐Detected Solid‐State NMR

## TL;DR

The paper introduces a new method using advanced NMR techniques to collect detailed structural data for proteins like bactofilin BacA.

## Contribution

The novel approach combines proton detection and 4D NMR techniques to efficiently obtain long-range distance restraints for protein structure determination.

## Key findings

- Several hundred unambiguous long-range distance restraints were collected for the core domain of bactofilin BacA.
- The method enables fast and reliable atomic-level structural investigations of proteins using solid-state NMR.
- The approach is applicable to a wide range of proteins, including newly discovered cytoskeletal proteins like bactofilin.

## Abstract

Solid‐state magic‐angle spinning NMR is a powerful technique for obtaining atomic‐resolution structural information of different types of biological macromolecules. The relatively low sensitivity and limited resolution of this method have continuously stimulated methodological developments to broaden its application range. In this study, we efficiently apply proton detection, moderate sample rotation, nonuniform sampling in four‐dimensional spectra, multiplexing, deuteration, and stereospecific labeling of methyl groups to collect several hundred unambiguous long‐range distance restraints based on proton–proton magnetization transfers. These data provide a reliable description of the core domain structure of full‐length bactofilin BacA, which belongs to a newly discovered class of cytoskeletal proteins. The work demonstrates the power of combining available techniques for fast and reliable atomic‐level structural investigations that can be applied to a wide range of proteins.

Proton detection, nonuniform sampling in 4D spectra, multiplexing, deuteration, and stereospecific labeling of methyl groups allowed for the collection of several hundred unambiguous long‐range distance restraints by solid‐state NMR for the rigid core of the bactofilin BacA.© 2026 WILEY‐VCH GmbH