Exploiting Anisotropic Orientation in Nanoparticle-Aided Cryo-Electron Microscopy Sampling.

Measuring the conformational distribution of small proteins is essential to understanding their role in biological systems. Multi-Tilt Nanoparticle-aided cryo-electron microscopy sampling (MT-NACS) was devised to measure the three-dimensional interparticle distance distribution (P(d)) of two gold nanoparticles (AuNPs) labeled on a protein by taking cryogenic electron microscopy (cryo-EM) images at multiple-tilt angles. However, tracking the same particles in a pseudo-tomographic manner during the multi-tilt cryo-EM experiments and data analysis requires extensive time and effort. Here, we report that proper incorporation of AuNP pair angle distribution allows reliable determination of the P(d) only using the cryo-EM images collected at a single tilt angle. The trends of structural changes in calmodulin (CaM) measured by single-tilt angle NACS (ST-NACS) and MT-NACS are consistent, as the tendencies of changes in the P(d) of AuNP-labeled CaM measured by both methods are similar. Our approach provides an efficient tool for measuring the conformational distribution and structural transition of small proteins.