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bioRxiv · 10.64898/2025.12.06.692658

Leveraging CryoEM and AI-Driven Morphological Feature Analysis for Insights on Bacterial Structures

Madugula, S. S.·Massenburg, L. N.·Brown, S. R.·Bible, A. N.·Harris, C. R.·Zhang, L. X.·Parker, K.·Retterer, S. T.·Morrell-Falvey, J. L.·Vasudevan, R. K.·Williams, A.
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Abstract

Bacteria adapt by undergoing dynamic structural changes in response to environmental cues, which are often indicative of deeper phenotypic shifts in physiology and behavior. Understanding these changes across length scales is crucial for elucidating bacterial lifecycles, informing antifouling surface design, enhancing pathogen detection, and advancing renewable energy applications. Cryogenic electron microscopy (cryoEM) enables high-resolution imaging of bacterial structures in hydrated, biologically relevant conditions. However, current quantitative analysis strategies that extract structural information from bacterial samples remain labor-intensive. This work presents an AI-driven segmentation workflow tailored to low-dose cryoEM datasets to rapidly analyze bacterial ultrastructural features from Pantoea sp. YR343, a Gram-negative bacterium isolated from the rhizosphere of Populus deltoides that forms robust biofilms along plant roots. YOLOv11 image segmentation quantifies inner and outer membrane thickness and flagella length, enabling automated analysis of bacterial ultrastructure. The workflow reliably distinguishes membranes from carbon edges of the TEM grid and contaminant crystalline ice while matching manual measurements and increasing throughput. Flagella detection routines additionally quantify nearest-neighbor proximity between bacterial envelopes and flagella. A field-of-view module further detects bacteria at low magnification for rapid screening. Together, these tools provide a scalable and automated framework for high-throughput, quantitative analysis of bacterial ultrastructure in cryoEM data. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=39 SRC="FIGDIR/small/692658v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@18eac2org.highwire.dtl.DTLVardef@1dc67a1org.highwire.dtl.DTLVardef@1179499org.highwire.dtl.DTLVardef@11d08f8_HPS_FORMAT_FIGEXP M_FIG AI-based tools enable rapid characterization of bacterial ultrastructure in low-dose cryoEM. The envelope thickness tool quantifies membrane thickness and anisotropy. The flagella module analyzes filament morphology and detects cell-flagella contacts. The field-of-view (FOV) module C_FIG

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Madugula, S. S., Massenburg, L. N., Brown, S. R., Bible, A. N., Harris, C. R., Zhang, L. X., Parker, K., Retterer, S. T., Morrell-Falvey, J. L., Vasudevan, R. K., Williams, A.. 2025-12-08. Leveraging CryoEM and AI-Driven Morphological Feature Analysis for Insights on Bacterial Structures. https://doi.org/10.64898/2025.12.06.692658

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