How to Choose the Right Method for Protein Structure Determination?
Proteins play essential roles in virtually all biological processes. Their functions, ranging from enzymatic catalysis and signal transduction to structural support within cells, are fundamentally dependent on their three-dimensional structures. However, amino acid sequences alone are insufficient to fully elucidate their biological functions. At the molecular level, protein structures not only reveal functional mechanisms but also provide atomic-level insights that enable applications such as drug target discovery, antibody engineering, and protein design. Several complementary techniques have been developed for protein structure determination, including X-ray crystallography, cryo-electron microscopy (Cryo-EM), nuclear magnetic resonance (NMR), and mass spectrometry (MS)-based approaches, each with specific advantages and limitations. Importantly, the selection of a suitable protein structure determination method is determined by the physicochemical properties of the sample, the biological question of interest, and the available experimental resources. Here, leveraging mass spectrometry expertise, we provide a systematic overview of the four major methodologies, their applicable scenarios, and technical features, aiming to facilitate rational decision-making in protein structure research.
X-Ray Crystallography: The High-Resolution Gold Standard
1. Suitable Scenarios
(1) Stable proteins with molecular weights >10 kDa
(2) Targets that can be successfully crystallized and require atomic-level structural accuracy
2. Highlights
(1) High resolution, often reaching 1 Å
(2) Well-suited for analyzing catalytic centers, ligand-binding pockets, and other critical structural motifs
3. Challenges
(1) Protein crystallization can be highly challenging
(2) Limited applicability for flexible or dynamic conformations
For stable, abundantly expressed proteins such as kinases or transporters, when atomic precision is required for structure-guided drug discovery, X-ray crystallography remains the preferred method.
Cryo-Electron Microscopy (Cryo-EM): A Tool for Large Macromolecular Complexes
1. Suitable Scenarios
(1) Large complexes (>200 kDa), including membrane proteins
(2) Flexible assemblies that are difficult to crystallize
2. Highlights
(1) Crystallization is unnecessary, and samples can be maintained in near-native states
(2) Advances in single-particle analysis continue to improve achievable resolution
3. Challenges
(1) High dependence on advanced instrumentation and sophisticated image processing
(2) Requires highly pure and aggregation-free samples
Cryo-EM is particularly suitable for investigating multi-protein assemblies, viral particles, and membrane-associated proteins, offering unique opportunities for studying complex systems.
Nuclear Magnetic Resonance (NMR): Preferred for Dynamics and Small Proteins
1. Suitable Scenarios
(1) Soluble proteins with molecular weights <40 kDa
(2) Studies of conformational dynamics, such as switching or ligand-induced structural changes
2. Highlights
(1) Measurements can be performed in solution, approximating physiological environments
(2) Provides detailed information on protein dynamics, supporting mechanistic investigations of regulation and function
3. Challenges
(1) Limited applicability to larger proteins
(2) Requires high protein concentrations (typically >0.5 mM)
NMR is well-suited for characterizing small proteins, structural domains, and conformational transitions, particularly during the early stages of mechanistic exploration.
Mass Spectrometry (MS)-Assisted Structural Biology: An Expanding Toolbox
Although not classified as a protein structure determination technique, mass spectrometry has emerged as a powerful complementary approach, particularly in probing conformational states, post-translational modifications, and protein interaction networks.
1. Crosslinking-MS
Captures spatial proximity between or within proteins through chemical crosslinkers, generating low-resolution distance constraints. This strategy is frequently applied to model macromolecular complexes and map protein-protein interaction interfaces.
2. Hydrogen-Deuterium Exchange-MS (HDX-MS)
Identifies flexible regions, conformational changes, and ligand-induced structural rearrangements, proving essential for studies of enzymatic mechanisms and antibody epitope mapping.
3. Structural Proteomics in Native Contexts
Combines proteomic techniques such as chaperone interactomics and transmembrane topology mapping to provide indirect evidence of structural states in situ.
By integrating advanced MS platforms (e.g., Orbitrap Fusion Lumos, timsTOF Pro 2) with specialized applications such as crosslinking-MS and HDX-MS, comprehensive perspectives can be achieved for structural analysis. Moreover, workflows that combine proteomics with structural-functional integration support the entire process from sample preparation to data interpretation, thereby accelerating functional studies and target validation. Each protein structure determination method offers unique advantages and limitations, and the optimal choice is not defined by which method is best, but rather by which is most appropriate for the specific scientific question. As structural biology advances, the strategic integration of complementary methods, particularly with the support of high-resolution mass spectrometry, provides powerful opportunities to advance protein science and therapeutic discovery.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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