Analyzing a protein’s structure involves examining its shape and folding across multiple levels using both experimental and computational tools. The process usually begins by purifying the protein sample, followed by techniques to reveal how the atoms are arranged.

• Laboratory methods like X-ray crystallography and cryo-electron microscopy (cryo-EM) are used to capture detailed 3D structures. X-ray crystallography requires the protein to form crystals, while cryo-EM freezes proteins in solution for imaging.

• Computational approaches, especially AI-based prediction models like AlphaFold, can infer likely 3D shapes from amino acid sequences.

• Spectroscopic tools such as circular dichroism (CD) help identify the presence of specific secondary structures like helices or sheets.

Together, these approaches provide insights into how the protein folds, how stable it is, and how it may interact with other molecules.

There are several methods tailored to understanding different levels of protein structure:

• Primary structure (amino acid sequence): Identified using mass spectrometry or sequencing.

• Secondary structure: CD spectroscopy and Fourier-transform infrared spectroscopy (FTIR) help detect common structural motifs like alpha-helices and beta-sheets.

• Tertiary and quaternary structures:
       ⊙ X-ray crystallography: Offers atomic resolution but requires protein crystallization.
       ⊙ Nuclear magnetic resonance (NMR): Works in solution but is best for smaller proteins.

• Cryo-EM: Ideal for large complexes and doesn’t require crystals.

• Cross-linking and mass spectrometry: Reveal spatial relationships between different parts of a protein.

• In silico modeling: Tools like homology modeling and machine learning-based predictions fill gaps where experimental data is lacking.

Often, a combination of these techniques provides the most complete picture.

Protein structure is categorized into four hierarchical levels, each identifiable using different methods:

• Primary structure (linear sequence of amino acids): Determined through sequencing or mass spectrometry.

• Secondary structure (local folding patterns): CD or infrared spectroscopy can estimate proportions of helices, sheets, and turns.

• Tertiary structure (3D folding of a single polypeptide): Solved through high-resolution tools like crystallography, NMR, or cryo-EM, or predicted computationally.

• Quaternary structure (multi-subunit assemblies): Studied via techniques that reveal subunit interactions, such as size exclusion chromatography, light scattering, or cryo-EM.