Annealing synchronizes the 70 S ribosome into a minimum-energy conformation

Abstract

Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.

Improving the Storage Stability of Soy Protein Isolate through Annealing

“…In particular, a 30 min annealing time corresponded to lower protein macromolecule content, reaching its lowest by the 12th week. This underscores the pivotal role of annealing duration in preserving protein structure [15].…”

Simulation of Complex Biomolecular Systems: The Ribosome Challenge

“…Such a raw data approach requires particular attention to all the details of the experiment that may affect the direct comparison. For example, the conformational ensemble of biomolecules captured by cryo-EM depends not only on the cryogenic temperature, but also on the temperature before cooling, as well as on the cooling rate (23,40,41,57,139,180). Thus, the conformational heterogeneity and the population of states observed in cryo-EM experiments do not necessarily reflect the room-temperature ensemble probed by simulations.…”

Autoinhibition and Activation Mechanisms for MICAL Monooxygenases in F-actin Disassembly

“…The freshly purified MICAL1 protein, with or without the treatment of GA-crosslinking, was concentrated to ~0.3 mg/ml for cryo-EM sample preparation. To improve conformational heterogeneity, the protein sample without GA-crosslinking underwent additional annealing in a 37°C water bath for 5 minutes, followed by rapid cooling on ice 52 . For sample preparation, 4 μL of the protein solution was injected onto a freshly glow-discharged grid (Quantifoil Cu, 300 mesh, 1.2/1.3) with a 5-second incubation followed by 3-second blotting with filter paper, and then quickly frozen in liquid ethane.…”