Z-Contrast Enhancement for Small Protein Cryo-EM Structure Determination

Abstract

Single-particle cryo-EM structure determination of small proteins has been challenging, which is mainly due to the poor signal-to-noise ratio (SNR) dictated by the low-dose imaging conditions required to avoid severe radiation damage. With the advent of direct electron detectors and phase plates, protein molecules with molecular weights (MWs) as low as 64 (hemoglobin [1]) and 52 (streptavidin [2]) kDa have recently been imaged and reconstructed to near-atomic resolution. These achievements indicate the potential of single-particle cryo-EM in structure determination of even smaller proteins. However, given the current state of phase-plate hardware and the associated control software development, optimal phase-plate utilization requires frequent conditioning and re-focusing during data collection, which is not yet suitable for routine, fully-automated large dataset acquisition. The electron scattering cross-section of an element depends on its atomic number (Z) and therefore differs substantially for heavier metal ions compared to the light atoms that are abundant in proteins (namely C, N, O). This physical property naturally endorses enhanced amplitude contrast in cryo-EM imaging of metalloproteins. Here we demonstrate that a copper storage protein [3] with a MW less than 50 kDa can be visualized by single-particle cryo-EM without the assistance of phase-plate. The electron density map has been reconstructed to 3.5 Å resolution upon multiset contrast transfer function correction. Beyond this work, the use of this Z-contrast enhancement approach has broader applications in single-particle EM structural biology.