Orienting the future of bio-macromolecular electron microscopy

With 40 years of development, bio-macromolecule cryo-electron microscopy（cryo-EM） has completed its revolution in terms of resolution and currently plays a highly important role in structural biology study. According to different specimen states, cryo-EM involves three specific techniques： single-particle analysis（SPA）, electron tomography and subtomogram averaging, and electron diffraction. None of these three techniques have realized their full potential for solving the structures of bio-macromolecules and therefore need additional development. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which include the air–water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimens, and electron radiation damage. Furthermore, potential solutions of these bottlenecks worthy of further investigation are proposed and discussed.

The determinant factors for map resolutions obtained using CryoEM single particle imaging method

The CryoEM single particle structure determination method has recently received broad attention in the field of structural biology. The structures can be resolved to near-atomic resolutions after model reconstructions from a large number of CryoEM images measuring molecules in different orientations. However, the determining factors for reconstructed map resolution need to be further explored. Here, we provide a theoretical framework in conjunction with numerical simulations to gauge the influence of several key factors to CryoEM map resolutions. If the projection image quality allows orientation assignment, then the number of measured projection images and the quality of each measurement(quantified using average signal-to-noise ratio) can be combined to a single factor, which is dominant to the resolution of reconstructed maps. Furthermore, the intrinsic thermal motion of molecules has significant effects on the resolution. These effects can be quantitatively summarized with an analytical formula that provides a theoretical guideline on structure resolutions for given experimental measurements.

A local-optimization refinement algorithm in single particle analysis for macromolecular complex with multiple rigid modules

Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macro- molecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate align- ments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orien- tations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimen- sional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large （60S/50S） and the small （40S/30S） subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolutionsingle particle analysis of macromolecular complexes with dynamic conformations.