Sub-50 kDa reconstruction with 2DTM

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.

Publication: Improved cryo-EM reconstruction of sub-50 kDa complexes using 2D template matching · eLife (2026).