Rucete ✏ Lehninger Principles of Biochemistry In a Nutshell
4.5 Determination of Protein and Biomolecular Structures
This chapter introduces the core experimental and computational methods used to determine the three-dimensional structures of proteins and other biomolecules, highlighting the strengths, applications, and process of each technique.
Structural Biology: Overview and Importance
• Structural biology uses physical, biochemical, and computational methods to determine the 3D structures of biomolecules (proteins, nucleic acids, membranes, carbohydrates).
• Knowing structure reveals function, molecular mechanisms, evolutionary relationships, and enables rational drug design and protein engineering.
• The three principal methods are x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM); methods are often combined for deeper insights.
• Computational tools (molecular dynamics, protein modeling, in silico folding) are increasingly important for predicting and analyzing structures, sometimes involving crowdsourced platforms like Rosetta@home and Foldit for protein design and folding simulations.
X-ray Crystallography
• X-ray crystallography determines atomic structure from diffraction patterns produced when x-rays pass through a well-ordered protein crystal.
• Each spot (reflection) in the diffraction pattern results from electrons in the atoms scattering x-rays; thousands of spots are recorded and analyzed computationally.
• Mathematical reconstruction (Fourier transform) generates an electron density map; atomic models are then built to fit this density.
• The method requires highly ordered crystals, which are difficult to obtain for some proteins (especially membrane and flexible proteins).
• Structures determined by crystallography represent a space- and time-averaged, functional conformation, though they lack information about molecular motion.
• X-ray analysis has solved over 100,000 protein structures, starting with myoglobin, revealing virtually all atomic positions except hydrogens.
Nuclear Magnetic Resonance (NMR) Spectroscopy
• NMR is performed on molecules in solution, providing dynamic information unavailable from crystals.
• Certain nuclei (e.g., 1H, 13C, 15N) produce NMR signals sensitive to chemical environment and spatial proximity.
• Multi-dimensional NMR (e.g., NOESY, TOCSY) provides distance and connectivity data between atoms, which is translated into distance constraints for structure calculation.
• Computer modeling assembles families of structures consistent with these constraints, reflecting molecular flexibility and motion.
• NMR is limited by protein size (works best for small to medium proteins), but isotopic labeling expands its reach.
Cryo-Electron Microscopy (Cryo-EM)
• Cryo-EM images thousands of individual molecules frozen in vitreous ice, avoiding damage and preserving native structure.
• Two-dimensional images of randomly oriented particles are computationally sorted and combined to produce high-resolution 3D structures.
• Particularly valuable for large, dynamic complexes, membrane proteins, and systems that resist crystallization or are too large for NMR.
• Cryo-EM has revealed structures of macromolecular machines (e.g., ribosomes, human telomerase) previously inaccessible by other means.
• Public resources like the EMDataResource archive 3D density maps and models for the scientific community.
Computational and Hybrid Methods
• Computational biochemistry (molecular modeling, dynamics) helps solve, refine, and predict structures, and enables in silico protein design (“designer proteins”).
• Crowdsourcing (Rosetta@home, Foldit) lets citizen scientists contribute to protein structure prediction and engineering, sometimes surpassing automated algorithms.
• Engineered proteins from computational designs have been synthesized, purified, and structurally validated by experimental methods.
In a Nutshell
• The structure of proteins and biomolecules can be determined by x-ray crystallography (from crystals), NMR spectroscopy (in solution), and cryo-EM (single particles in ice), each with unique advantages.
• Computational and crowdsourced methods are increasingly essential, both for modeling and for creating proteins with novel functions.
• Understanding structure is fundamental for exploring molecular function, mechanism, and designing new biomolecules for research and medicine.