A2 - Resolving structural changes in 4D with PELDOR: CRISPR/Cas13a
Clustered regularly interspaced short palindromic repeats associated endoribonucleases (CRISPR/Cas) are adaptive immune systems of bacteria and archaea. Several variations have been identified, among them the CRISPR/Cas9 system widely used in eukaryotic gene editing and therapy. Here, we propose to study how the related, less-investigated CRISPR/Cas13a system works in precise time-resolved molecular terms. To this end, we will use our recently developed time-resolved Pulsed Electron-Electron Double Resonance (trPELDOR) spectroscopy in combination with fully atomistic molecular dynamics (MD) simulations. In the methodological part, we aim at 1) reducing the deadtime of trPELDOR below 80 µs to be able to resolve faster structural changes and 2) using the MD trajectories to fit the structures of the complexes to the trPELDOR time traces by minimizing the differences between simulated and measured time trace. This combined approach will enable us to not only follow the dynamics of structural changes but also to elucidate molecular driving forces and relate them to functional causes and effects.
Figure 1: a) Structure of the Lbu crRNA/Cas13a complex (PDB: 5XWY) with MTSSL rotamers at sites A462/S660 modelled into it with mtsslWizard (red sticks). b) PELDOR time traces of this variant in its apo- (black), precrRNA (blue) and crRNA (yellow) bound state. The red lines are the fits to the data. c) The corresponding distance distributions with the same color code as in (b) overlayed with the predicted distance distributions for the apoCas13a (green area) and crRNA/Cas13a (orange area) states. These distributions were calculated with mtsslWizard using the Alpha-Fold generated apoCas13a and the crystal structure of crRNA/Cas13a as input structures.
Figure 2: Morph of Cas13a from the ligand-free structure (AlphaFold) via the CRISPR-bound state (PDB-ID: 5xwy, cryo-EM) to the CRISPR/target RNA bound state (PDB-ID: 5xwp, X-Ray).