Instruct-ULTRA D7.2 Standardised workflows for integrative sample preparation methods for structural biology in cells

Performing structural studies in a cellular context provides a view and understanding of proteins that cannot be obtained on purified samples in simple aqueous environments. Interactions with cellular partners can be measured in the crowded, natural cellular environment that strongly influences the dynamics, affinities and kinetics of proteins and their interactions. Proteins also span a spectrum of foldedness, from well-folded to intrinsically disordered, and these states are also affected strongly by the cellular milieu.

In the first NMR-focused part of this work package, partner P12 (CIRMMP) developed and optimise d an NMR bioreactor system to standardise the workflow of real-time in-cell NMR applications. Different configurations were tested for supporting human cells in biocompatible hydrogels. A workflow was optimised, allowing up to ~72 hours of NMR experiments with high cell viability, and applied to observe real-time protein-ligand interactions. P10 (CNRS Grenoble) extended the field of application of in-cell NMR to large proteins (a challenging area for NMR) using 13CH3 specific isotopic labelling. They successfully demonstrated this strategy on an 82 kDa target in bacterial cells and developed new 13CH3-labelling protocols to extend its application to eukaryotic cells at the limit of this method. P13 (UU) also developed labelling approaches, for parallel investigations of macromolecular complexes involving intrinsically disordered proteins, by NMR and small angle scattering (SAS) in vitro and for studies of these complexes by in-cell NMR. They established a workflow permitting collection, from the same sample, of both local structural information by NMR and overall shape information (demonstrated on a 120 kDa IDP-complex). This was further extended to interactions of IDPs by in-cell NMR.In the second focus of this work package, P10 (CNRS Strasbourg) established a workflow based on an in-cell CRISPR/Cas9 methodology. The methodology permits purification of macromolecular complexes directly from endogenous sources, and labelling of individual proteins and macromolecular complexes with fluorescent reporter tags for imaging applications in both electron and light microscopy.