Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra

Abstract

Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6–10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.

Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding

“…No anomalous scattering signal was detected in the second EF-hand loop, but electron density consistent with the coordination of a sodium ion was present (Figure 1, panel c). While attempts to crystallize EH1.2 proved unsuccessful, and as part of an integrative structural biology approach, we obtained structural insights for both EH1.1 (PDB: 6YEU) and EH1.2 (PDB: 6YET), in solution, by NMR (Evangelidis et al, 2018). With respect to EH1.2, the NMR and X-ray structures agree very well with an RMSD of 1Å (compared by the Dali algorithm ( (Holm, 2019)).…”

“…For each protein a set of three sparsely sampled 4D NMR experiments was acquired: 4D HC(CC-TOCSY(CO))NH, 4D 13 C, 15 N edited HMQC-NOESY-HSQC (HCNH), and 4D 13 C, 13 C edited HMQC-NOESY-HSQC (HCCH). Sequential and aliphatic side chain assignments were obtained automatically using the 4D-CHAINS algorithm that combines through-bond information from the 4D-TOCSY experiment and distance information from the 4D-NOESY (HCNH) experiment (Evangelidis et al, 2018). Aromatic sidechain frequencies were assigned manually by recording an additional 3D 13 C edited NOESY-HSQC experiment.…”

Hybrid Thermophilic/Mesophilic Enzymes Reveal a Role for Conformational Disorder in Regulation of Bacterial Enzyme I

“…Similar notation will be used for the full-length enzymes (eEI and tEI, respectively). eEI and tEI share a similar sequence (overall identity 54%; EIC identity 60%; active site identity 100%, Supplementary Figure S1) and 3D structure [20,[29][30][31]. While the temperature dependence of eEI and tEI activities has not been investigated yet, the E. coli and T. tengcongensis PTS's were shown to be optimally active at 37 °C and 65 °C, respectively [30].…”

A Practical Perspective on the Roles of Solution NMR Spectroscopy in Drug Discovery

“…Despite its application in determining structures of small-molecular-weight compounds, NMR is still a powerful tool to resolve structures of macromolecules such as proteins and DNA/RNA [22,99]. The newly developed methods make it possible to determine the structures of protein in a short period of time [100]. There are numerous structures resolved by NMR every year, providing valuable insights into structure-based drug design.…”