Apoferritin Double-Shell Nanostructures for Small-Protein Cryo-EM

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Solution Overview

Problem

Current methods in cryogenic-electron microscopy (cryo-EM) struggle to resolve protein structures below 40 kDa with a resolution better than 4 Å, limiting the structural analysis of small and flexible proteins.

Innovation Solution

Protein double-shell nanostructures comprising apoferritin are used to increase the rigidity of cargo proteins, allowing for improved resolution in cryo-EM by incorporating a cargo protein connected to apoferritin and a tag protein, which enhances structural clarity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If cryo-EM is applied to small proteins below 50 kDa, then the structural analysis capability is extended to smaller proteins, but the resolution deteriorates to worse than 4 Å for proteins below 40 kDa

Engineering Contradiction:

Improvestructural analysis capabilityVSAvoidresolution

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The patent embeds small cargo proteins (below 40 kDa) inside a nested structure consisting of an inner apoferritin shell and an outer tag protein shell. This nested doll approach allows the small protein to be housed within a larger, more rigid composite structure that provides the necessary structural stability for high-resolution cryo-EM imaging while preserving the cargo protein's native conformation and function.

Inventive Principle:

Principle #7Nested doll (Nesting)

Solution Approach 2:

The patent creates a composite nanostructure by combining apoferritin (inner shell) with tag proteins (outer shell) to form a hybrid structure. This composite material approach leverages the strengths of both components: apoferritin provides a rigid cage structure, while the tag protein layer adds further structural support and surface properties, collectively enabling high-resolution imaging of the embedded small protein.

Inventive Principle:

Principle #40Composite materials

2Reliability

If small and flexible proteins are imaged directly by cryo-EM, then the native state is preserved, but the structural clarity deteriorates due to flexibility and small size

Engineering Contradiction:

Improvenative state preservationVSAvoidstructural clarity

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent applies local quality by providing rigidification only to the specific regions where structural support is needed (at the interfaces between cargo protein and apoferritin, and between tag protein and cargo protein) while leaving the core cargo protein structure untouched. This localized approach maintains the native state of the cargo protein while improving overall structural clarity through strategic rigidification at boundary regions.

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20250376503A2Protein double-shell nanostructures and their use

Publication Date: 2025.12.11 THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV

AI summary

Protein double-shell nanostructures comprising apoferritin for carrying cargo proteins of interest are provided. Such nanostructures can be used to increase rigidity of a cargo protein of interest to allow structures of small and flexible proteins to be determined by cryogenic-electron microscopy (cryo-EM). Recombinant vectors for producing protein double-shell nanostructures are also provided. The nanostructures described herein may find use in various applications in research and drug discovery.