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Area of Science:

  • Biophysics
  • Structural Biology
  • Materials Science

Background:

  • Tubulin assembles into 2D sheets in the presence of zinc, serving as a model for tubulin and microtubule structure.
  • These sheets are ideal for atomic force microscopy (AFM) due to their 2D crystalline lattice and retained structural features.

Purpose of the Study:

  • To evaluate common scanning probe microscopy substrates for immobilizing tubulin sheets.
  • To determine how substrate interactions affect tubulin sheet morphology and adsorbate contamination.
  • To identify optimal substrates for high-resolution AFM imaging of tubulin sheets.

Main Methods:

  • Tubulin sheets were adsorbed onto mica, gold, highly ordered pyrolytic graphite (HOPG), and carbon-coated electron microscopy (EM) grids.
  • Atomic force microscopy (AFM) was used to image the immobilized tubulin sheets on each substrate.
  • Structural characterization and assessment of adsorbate contamination were performed for each substrate.

Main Results:

  • Tubulin sheets were successfully imaged on all tested substrates.
  • Carbon-coated EM grids yielded the most consistent results, preserving fine structural features.
  • Carbon-coated EM grids exhibited the least contamination from unpolymerized tubulin adsorption.
  • AFM images favorably compared with published electron micrographs.

Conclusions:

  • Substrate choice is critical for nonperturbative immobilization and high-resolution imaging of 2D protein crystals like tubulin sheets.
  • Carbon-coated EM grids are identified as a suitable substrate for AFM studies of tubulin sheets.
  • This study provides a foundation for future investigations into tubulin and microtubule structure using AFM.