Structural Analysis of Hierarchical Protein Assemblies

Structural Analysis of Hierarchical Protein Assemblies

  • Course: BIOS6120 Structure, Function and Analysis

  • Instructor: Dr. Wei-Feng Xue

  • Term: Autumn 2025

Overview of the Two Lectures

  • Length scales in Biology

  • Overview of methods for structural determination

  • Key points on scattering techniques

  • Introduction to microscopy methods

  • Atomic Force Microscopy (AFM)

  • Electron Microscopy

  • Data analysis and modeling methods

  • Integrative approaches in structural analysis

Key 3D Structural Analysis Methods

  • X-ray Crystallography:

    • Resolves structures from ~100 nm to 0.1 nm.

  • NMR Spectroscopy:

    • Resolves structures from ~10 nm to 0.1 nm.

  • Scattering Techniques:

    • Static Light Scattering

    • Dynamic Light Scattering

    • Small Angle X-ray/Neutron Scattering: from ~100 nm to 1 nm.

  • Microscopy (and Nanoscopy):

    • Optical (Transmission) Microscopy: > ~1000 nm.

    • Fluorescence Microscopy: > ~1000 nm.

    • Electron Microscopy: ~10,000 nm to 0.3 nm.

    • Scanning Probe Microscopy: including AFM, resolving from ~10,000 nm to 1 nm.

Length Scales in Biology

  • Common conversions of measurements:

    • 1 metre (m) ≈ 3.28 feet ≈ 39.37 inches.

    • 1 micro-metre (μm) = 1 millionth (1/1,000,000) of a metre.

    • 1 nano-metre (nm) = 1 billionth (1/1,000,000,000) of a metre.

  • Diagram representation of lengths in biology:

    • 1 nm, 10 nm, 100 nm, 1 μm, 50 μm, with respective micro and millimetre conversions.

Small Angle Scattering Techniques

  • Advantages:

    • No Need for Crystals: Scattering does not require crystalline samples or special preparation.

    • Molecular Mass Independence: Applicable under nearly physiological conditions.

    • Quantitative Analysis: Allows for comprehensive structural analysis of complex biological systems.

    • Speed: Facilitates high throughput, screening, and real-time analysis.

    • Structure Resolution: Operates within an overall structure scale of ~100 nm to 1 nm.

    • Sample Requirements: Requires large quantities and high concentrations of samples.

    • Complementarity: Works well alongside NMR techniques.

SAXS Structural Analysis with "Bead Models"

  • Detailed illustration of the scattering intensity as a function of distance distribution with example bead models (spherical, prolate, oblate, etc.).

Atomic Force Microscopy (AFM)

  • AFM is categorized as a type of scanning probe microscopy.

    • Its original scanning tunneling microscope was invented by Gerd Binnig and Heinrich Rohrer, awarded the Nobel Prize in Physics in 1986.

    • Applications include imaging DNA, proteins, virus particles, membranes, and whole cells.

    • AFM became commercially available in the late 1980s to 1990s.

Examples of AFM Applications

  • Functional Capabilities:

    • Operates in both air and fluid.

    • Modes: Contact mode, tapping mode, and force-distance mode.

    • Useful for high-resolution imaging, size distribution analysis, and surface structure assessment.

    • Applicable to biological samples: proteins, nucleic acids (DNA/RNA), membranes, and cells.

Image and Data Management in AFM

  • AFM technique overview: detail of device operation, including protocols for powering the device and orientation.

  • Force-Distance Relationship:

    • Graphical representation of force vs. distance illustrating approach and withdrawal phases during AFM engagement.

  • True 3D topology creation showing sample surface features and analysis protocols.

Electron Microscopy Overview

  • Mounts on electron beams that exhibit significantly shorter wavelengths than light beams.

  • First developed by Ernst Ruska; awarded the Nobel Prize in Physics for his foundational work.

  • Applications focus on biological entities like protein assemblies and cells.

Types of Electron Microscopy

  • Scanning Electron Microscopy (SEM): Involves coating specimens in heavy metals.

  • Transmission Electron Microscopy (TEM): Focuses on specimen sections; can also include negative staining methods.

  • Scanning Transmission Electron Microscopy (STEM): Suitable for quantitative mass determination of samples.

Cryogenic-TEM (cryo-EM)

  • Suitable for determining 3D structures of large proteins and assemblies (>500 kDa).

  • Data capture occurs at temperatures < -160°C to prevent ice crystal formation.

  • Advantages include preservation of structure due to rapid freezing and protection against fragmentation.

  • Results in lower image contrast and dictates additional processing.

Structural Modeling Approaches

  • Tomography: Images a single specimen from different angles to reconstruct 3D visualizations.

  • Single Particle Analysis: Includes methods for classifying and averaging various orientations of particles, essential for determining the 3D structure.

Case Study: Amyloid Fibrils

  • Associated with several diseases:

    • Type II diabetes mellitus, Alzheimer’s disease, Parkinson’s disease, etc.

    • Structures can range broadly in scale, typically ~10 nm in width and several microns in length.

AFM vs. TEM Comparison

  • Critical differences in data acquisition:

    • AFM yields true 3D topological maps, operates under various conditions, and requires no staining.

    • TEM provides high-resolution images at lower signal-to-noise ratios, often necessitating chemical staining.

Combined Approaches for Amyloid Fibrils

  • Integration of multiple techniques such as AFM, NMR, X-ray diffraction, and cryo-EM as outlined in recent studies demonstrates a layered analysis framework for understanding amyloid structure.

Integrative Approaches Linking AFM and Cryo-EM

  • Methods to link data collected by both technologies, enhancing protein structure models using 3D data derived from AFM in conjunction with cryo-EM techniques.

Recommended Readings

  • “How Proteins Work” by Mike Williamson: Chapters 11.5, with additional examples in Chapters 9 and 10.

  • Notable papers on electron microscopy advancements and developmental studies in cryo-EM and AFM applications.

  • Example citations provided for relevant literature to explore further.