Table 1.

F-actin–ABP structural determination techniques

Technique	Advantages	Limitations
X-ray crystallography	Atomic resolution Suitable for G-actin	No 3D crystal structures for F-actin–ABP complexes
NMR	Atomic resolution Dynamic information	Large sample amounts, protein labeling, >40 kDa difficult
Electron crystallography	nm to atomic resolution	Well ordered 2D crystals, technically difficult
Electron microscopy	Study of large complexes like F-actin Study interactions between actin monomers	Electron dense staining and UHV, resolution limit ~ 5 Å
Cryo-EM	3.5–4 Å resolution More informative ABP–F-actin complex architecture Multiple orientations of ABP–F-actin complexes	<100 kDa difficult Not suitable for analyzing ABP induced changes in individual filaments
Atomic force microscopy	μm to atomic resolution No fixing, electron dense staining, UHV Quantitative 3D structural information on single ABP–F-actin complexes Study of ABP–F-actin structure heterogeneity by measuring individual helix without averaging Measurement of nanoscale assembly of ABPs on F-actin for binding cooperativity determination Inexpensive equipment compared to the above techniques; simple instrumentation design and data interpretation	Surface technique Immobilization of ABP–F-actin on substrates Relatively new tool in ABP–F-actin structural studies
Light microscopy/FRET/TIRF	Dynamic studies mm to ~300 nm resolution range Most commonly used technique for ABP–F- actin binding cooperativity determination	Diffraction limit ~ 200–300 nm