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Enhancing Density Maps by Removing the Majority of Particles in Single Particle Cryogenic Electron Microscopy Final Stacks

Over the past decade, advancements in technology and methodology within the field of cryogenic electron microscopy (cryo-EM) single-particle analysis ...

Yau Mathematical Sciences Center

Yanqi Lake Beijing Institute of Mathematical Sciences and Applications

State Key Laboratory of Membrane Biology, School of Life Science

The structural variations of multidomain proteins with flexible parts mediate many biological processes, and a structure ensemble can be determined by selecting a weighted combination of representative structures from a simulated structure pool, producing the best fit to experimental constraints such as interatomic distance. In this study, a hybrid structure-based and physics-based atomistic force field with an efficient sampling strategy is adopted to simulate a model di-domain protein against experimental paramagnetic relaxation enhancement (PRE) data that correspond to distance constraints. The molecular dynamics simulations produce a wide range of conformations depicted on a protein energy landscape. Subsequently, a conformational ensemble recovered with low-energy structures and the minimum-size restraint is identified in good agreement with experimental PRE rates, and the result is also supported by chemical shift perturbations and small-angle X-ray scattering data. It is illustrated that the regularizations of energy and ensemble-size prevent an arbitrary interpretation of protein conformations. Moreover, energy is found to serve as a critical control to refine the structure pool and prevent data overfitting, because the absence of energy regularization exposes ensemble construction to the noise from high-energy structures and causes a more ambiguous representation of protein conformations. Finally, we perform structure-ensemble optimizations with a topology-based structure pool, to enhance the understanding on the ensemble results from different sources of pool candidates.

Investigating energy-based pool structure selection in the structure ensemble modeling with experimental distance constraints: The example from a multidomain protein Pub1.

Cryogenic electron microscopy (cryo-EM) is widely used to determine near-atomic resolution structures of biological macromolecules. Due to the low signal-to-noise ratio, cryo-EM relies on averaging many images. However, a crucial question in the field of cryo-EM remains unanswered: how close can we get to the minimum number of particles required to reach a specific resolution in practice? The absence of an answer to this question has impeded progress in understanding sample behavior and the performance of sample preparation methods. To address this issue, we develop an iterative particle sorting and/or sieving method called CryoSieve. Extensive experiments demonstrate that CryoSieve outperforms other cryo-EM particle sorting algorithms, revealing that most particles are unnecessary in final stacks. The minority of particles remaining in the final stacks yield superior high-resolution amplitude in reconstructed density maps. For some datasets, the size of the finest subset approaches the theoretical limit.

Article	Year
Investigating energy-based pool structure selection in the structure ensemble modeling with experimental distance constraints: The example from a multidomain protein Pub1. Proteins\| PubMed ID: 29383828	2018
A minority of final stacks yields superior amplitude in single-particle cryo-EM. Nature communications\| PubMed ID: 38072910	2023

Chenglong Bao

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