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Authors include: Wang Tongtong, Li Zhangqiang (Tsinghua University School of Life Sciences and School of Medicine), Xu Kun (Research Fellow in Computational Biology at Tsinghua University, focusing on deep learning and its applications in the real world and the microscopic world), Huang Wenzhe (Tsinghua University School of Life Sciences), Huang Gaoxingyu (Westlake Institute for Advanced Study, Department of Biology), Zhang Qiangfeng (Tsinghua University School of Life Sciences, Center for Life Sciences, research involving structural biology, genomics, artificial intelligence, and big data analysis), Yan Ning (Shenzhen Institute of Medical Sciences, Shenzhen Bay Laboratory, Tsinghua University, research focusing on the structure and function of membrane proteins).

CryoSeek: A Strategy for Discovering Bioentities using Cryo-Electron Microscopy

Abstract

• The breakthroughs in cryo-electron microscopy (cryo-EM) and AI-assisted structure prediction offer opportunities to explore unknown bioentities.
• CryoSeek strategy is proposed, which involves direct analysis of natural environmental samples using cryo-EM to discover unknown bioentities.
• The concept was validated using water samples from the Hetao Pond at Tsinghua University, successfully reconstructing cryo-EM maps of two unknown fibrous structures.

Introduction

• Traditional structural biology has focused on known bioentities.
• Advances in cryo-EM and AI technologies now make it possible to directly explore unknown bioentities.
• CryoSeek strategy: Discovering unknown bioentities from natural environmental samples.

Methods

• Collected water samples from the Hetao Pond at Tsinghua University.
• Simply filtered and concentrated the water samples.
• Acquired cryo-EM data.
• Used CryoNet to automatically build models.
• Conducted structure and sequence alignment analysis.

Results

• Reconstructed cryo-EM maps of two unknown fibrous structures, TLP-1a and TLP-1b.
• TLP-1a/b structures are novel and originate from uncharacterized proteins of unknown species.
• CryoSeek strategy shows potential for application in samples from extreme environments.

Discussion

• CryoSeek brings a paradigm shift to structural biology.
• High-resolution structure determination can guide other characterization methods to explore unknown bioentities.
• CryoSeek can be extended to other biomes, promoting the development of structural X-ology.

Materials and Methods

• Sample preprocessing.
• Cryo-EM sample preparation and data acquisition.
• Data processing.
• Model building and optimization.
• Metagenomic sequencing and data analysis.
• Mass spectrometry analysis and data interpretation.

Data and Software Availability

• Cryo-EM maps and atomic coordinates have been archived.
• All other data are included in the manuscript and supplementary materials.

Q: What specific research methods were used in the paper?

• Sample collection: Water samples were collected from the lotus pond at Tsinghua University.
• Sample preprocessing: The water samples were filtered and concentrated.
• Cryo-EM analysis: Cryo-electron microscopy imaging was performed on the preprocessed samples.
• Data processing: cryoSPARC software was used for 2D classification, 3D reconstruction, and refinement.
• Model building: CryoNet software was used to automatically build atomic models.
• Structural comparison: Foldseek was used to search for structures similar to TLP-1a/b.
• Sequence analysis: BLAST analysis was conducted on the sequences of TLP-1a/b.
• Bioinformatics analysis: Metagenomic sequencing and mass spectrometry were performed on the samples.

Q: What are the main research findings and achievements?

• Two unknown protein fiber structures, named TLP-1a and TLP-1b, were discovered.
• Atomic models of TLP-1a and TLP-1b were successfully constructed using CryoNet software.
• Structural comparison revealed that TLP-1a/b share structural similarities with bacterial flagella.
• The CryoSeek strategy was proposed, which involves using cryo-EM to directly explore unknown biological entities.

Q: What are the current limitations of this research?

• The species origin of the unknown biological entities has not yet been determined.
• Mass spectrometry and metagenomic sequencing failed to find matching sequences for TLP-1a/b, possibly due to low protein content or incomplete sequence database coverage.
• The CryoSeek strategy may have limitations when dealing with highly heterogeneous samples, such as those collected from extreme environments.
• The current research is limited to laboratory conditions and has not been extended to a wider range of environments or spaces.