Improved cryo-EM reconstruction of sub-50 kDa complexes using 2D template matching
← All research projectsView figure ↗A clearer view of protein side chains and ATP in a ~41 kDa protein kinase A complex, comparing the conventional single-particle map, the 2DTM template, and the 2DTM reconstruction.
We extend single-particle cryo-EM to the sub-50 kDa regime by leveraging 2D template matching (2DTM) for accurate particle alignment and stringent particle selection. Applying our method to a previously published ~41 kDa catalytic domain of protein kinase A dataset, we demonstrate improved visualization of ligands and binding pockets compared to traditional workflows. We further present calculations suggesting that the molecular-weight limit can be pushed below 10 kDa with the combined use of a phase plate and liquid-helium cooling.
Building a composite omit map. We systematically delete different residues throughout the protein, run 2DTM with each omit template, keep only the density near the deleted atoms, and assemble the pieces into a map free of template bias.
