Effects of cryo-EM cooling on structural ensembles

Abstract

Structure determination by cryo electron microscopy (cryo-EM) provides information on structural heterogeneity and ensembles at atomic resolution. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to cryogenic temperatures. To what extent the structural ensemble is perturbed during cooling is currently unknown. Here, to quantify the effects of cooling, we combined continuum model calculations of the temperature drop, molecular dynamics simulations of a ribosome complex before and during cooling with kinetic models. Our results suggest that three effects markedly contribute to the narrowing of the structural ensembles: thermal contraction, reduced thermal motion within local potential wells, and the equilibration into lower free-energy conformations by overcoming separating free-energy barriers. During cooling, barrier heights below 10 kJ/mol were found to be overcome, which is expected to reduce B-factors in ensembles imaged by cryo-EM. Our approach now enables the quantification of the heterogeneity of room-temperature ensembles from cryo-EM structures.

Electron microscopy analysis of soft materials with freeze‐fracture techniques

“…Structural analysis of nano‐ and micro‐materials is essential in various material sciences such as polymers, 1–4 energy materials, 5,6 semiconducting materials, 7–9 and biomaterials 10–16 . Depending on the size and properties of the material, sophisticated pretreatment, and observation techniques are essentially required.…”

“…Structural analysis of nano-and micro-materials is essential in various material sciences such as polymers, [1][2][3][4] energy materials, 5,6 semiconducting materials, [7][8][9] and biomaterials. [10][11][12][13][14][15][16] Depending on the size and properties of the material, sophisticated pretreatment, and observation techniques are essentially required. Scanning electron microscopy (SEM), 2,17,18 transmission electron microscopy (TEM), 11,[19][20][21][22] atomic force microscopy (AFM), [23][24][25] and x-ray diffraction (XRD) [26][27][28] are generally used to analyze structures that have nanometer scales.…”

Triggered functional dynamics of AsLOV2 by time-resolved electron paramagnetic resonance at high magnetic fields

“…However, most state-of-the-art structural biology tools require the protein to be immobilized (cryo-electron microscopy, solid-state NMR, or double electron electron resonance – DEER) or mechanically inhibited (X-ray crystallography) which may cause a significant amount of information to be lost ( e.g ., time-dependence, environmental effects, pH effects). [2, 3] By rapidly freezing an ensemble of proteins after triggering a conformational change, several structural biology tools have been used to elucidate the triggered functional dynamics of proteins [4–6]. However, ideally, a “movie” would be “filmed” in real-time, by in vitro tracking of the positions of residues at or near physiological temperatures in the solution state.…”

In-Vitro Diagnostic Reagent Evaluation of Commercially Available Cardiac Troponin I Assay Kits Using H/D Exchange Mass Spectrometry for Antibody-Epitope Mapping

“…Moreover, high resolution is obtained only at low temperatures. 19 In contrast, commercial HDX-MS experimental devices and their data processing software are highly convenient to use. 17 Epitope analysis in such devices is performed in solution, which allows various environmental conditions to maximize the simulations of analyte interactions.…”

“…Even though it is suitable for the in-depth analysis of essential proteins, Cryo-EM requires considerable human and computational resources to process large amounts of data. Moreover, high resolution is obtained only at low temperatures . In contrast, commercial HDX-MS experimental devices and their data processing software are highly convenient to use .…”