# CryoEM Structures of LF-Anthrax pore translocon at pH 5.5

> **NIH NIH R03** · UNIVERSITY OF KANSAS MEDICAL CENTER · 2020 · $73,850

## Abstract

Project Summary/Abstract
The primary goal of this project is to use cryo-EM single particle analysis to obtain atomic resolution
structures (2.2-2.9 Å) of the functional lethal anthrax toxin containing the N terminal domain lethal factor (LFN)
bound to the anthrax toxin protective antigen (PA) pore complexes inserted into POPC lipid nanodiscs (LFN-PA
pore-nanodiscs). This complex, as well as the PA-pore nanodisc complex, are exposed to translocation
competent pH conditions (5.5) and the atomic structures of each of these single LFN or 3 bound LFN to the
anthrax pore will be evaluated to uncover the dynamic differences between translocation competent (pH 5.0).
These forms will be compared with the inhibited (pH 7.5) forms. Our preliminary structure with a single data
collection session on Hong Zhou's Titan Krios yielded a 5.9 A structure that appears to show extra density from
the LFN unstructured tail associated with the phe clamp pore lumen. More high resolution data is required to
obtain an atomic resolution structures will be obtained from in collaboration with Hong Zhou group (UCLA)
(prescreening with Tommi White- MU). We routinely obtain superior solubilized nanodisc (lipid bilayer)
complexes in vitreous ice of both 1LFN-PA pore and saturated 3LFN-PA pore complexes to at pH 5.5 enabling
us to observe the pH induced changes in atomic structures of these PA pore complexes. Our previously NIH
R01 funded structural analysis with an CCD detector (Wah Chiu NCMI site Baylor) culminated in a complete 17
Å 3:1 LFN-PA pore nanodisc complex structure. Acquisition of the desired molecular detail of these initial
engagement complexes will specifically provide the atomic snapshots of complexes in various physiologically
relevant pH environments.
Research and Biotechnology implications:
 • Our novel methods to generate cryo-EM ready samples and acquire atomic structure of the PA pore
 translocon nanodisc complexes should serve as a reasonable template to enhance the acquisition of
 atomic structure for other aggregation prone membrane proteins inserted into authentic lipid bilayers.
 • Our approaches allow us to both control initial ligand association complexes for easier sample
 preparation of 1:1 and 3:1 (LFN:PA pore) complexes and specific population selection methodologies
 for cryo-EM analysis. Our research represents the first successes in constructing late endosomal
 anthrax complexes at pH 5.0 using novel solution protocols. Our approach is highly relevant toward
 reconstructing other bacterial toxin and viral interactions under late endosome conditions.
 • These easy yet powerful approaches of controlled immobilized construction and release are most
 certainly applicable toward other problem protein constructions where pH dependent changes occur
 that result in membrane insertion that dictate bacterial toxin and viral entry into the cell.

## Key facts

- **NIH application ID:** 9843485
- **Project number:** 5R03AI142361-02
- **Recipient organization:** UNIVERSITY OF KANSAS MEDICAL CENTER
- **Principal Investigator:** Bret D Freudenthal
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $73,850
- **Award type:** 5
- **Project period:** 2019-01-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9843485

## Citation

> US National Institutes of Health, RePORTER application 9843485, CryoEM Structures of LF-Anthrax pore translocon at pH 5.5 (5R03AI142361-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9843485. Licensed CC0.

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