12.1 Protein crystallography and structure determination
4 min read•august 16, 2024
Protein crystallography is a powerful technique for determining the 3D structure of proteins at atomic resolution. It involves growing protein crystals, exposing them to X-rays, and analyzing diffraction patterns to reconstruct the structure.
This method is crucial in biology and pharmaceuticals, allowing scientists to understand protein function, design drugs, and study disease mechanisms. It combines physics, chemistry, and biology to unlock the secrets of life's molecular machinery.
Protein Crystallography Principles
Fundamentals of X-ray Crystallography
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- Protein crystallography determines three-dimensional protein structures at atomic resolution using
- Process involves growing protein crystals, exposing them to X-ray beams, and analyzing diffraction patterns to reconstruct structure
- () relates X-ray wavelength to crystal plane spacing and diffraction angle
- Phase information crucial for structure determination lost during data collection leads to ""
- Methods to solve phase problem include , , and techniques
- Molecular replacement uses known structures of similar proteins
- Isomorphous replacement introduces heavy atoms into the crystal
- Anomalous dispersion exploits anomalous scattering of certain atoms
Advanced Techniques and Instrumentation
- sources provide high-intensity X-ray beams
- Allow faster data collection and higher resolution structures (up to ~1 Å)
- Examples include Advanced Photon Source (USA) and Diamond Light Source (UK)
- Cryo-crystallography techniques reduce radiation damage and improve data quality
- Involve flash-freezing crystals in liquid nitrogen (temperature ~100 K)
- (glycerol, ethylene glycol) prevent ice formation
- enable serial femtosecond crystallography
- Allow study of radiation-sensitive proteins and time-resolved experiments
Growing Protein Crystals
Protein Purification and Preparation
- High-purity protein crucial for crystal growth obtained through chromatography techniques
- Examples include affinity chromatography, ion exchange, and size exclusion
- Assess protein purity using methods like SDS-PAGE and mass spectrometry
- Crystallization occurs when protein solution reaches supersaturated state
- Achieved through controlled precipitation methods
- Balance between protein-protein and protein-solvent interactions
Crystallization Techniques and Optimization
- Common crystallization techniques include , , and
- Vapor diffusion methods (hanging drop, sitting drop) most widely used
- Screen crystallization conditions by varying parameters
- Protein concentration (typically 5-20 mg/mL)
- pH (range 4-9)
- Temperature (4°C, 20°C common)
- Precipitants (PEG, ammonium sulfate, MPD)
- promote crystal growth or improve quality
- Microseeding introduces small crystal fragments
- Macroseeding uses larger, pre-existing crystals
- Assess crystal quality based on size, shape, and diffraction properties
- Larger, single crystals generally more suitable for X-ray diffraction
- Ideal crystal size ranges from 0.1-0.5 mm
- Post-crystallization treatments improve diffraction quality or facilitate structure determination
- Dehydration can tighten crystal packing
- Soaking with heavy atoms aids phase determination
- Ligand soaking allows study of protein-ligand complexes
Analyzing Diffraction Data
Data Collection and Processing
- Record diffraction patterns at various crystal orientations using rotating crystal method
- Integrate diffraction spots and scale intensities to obtain complete dataset of amplitudes
- derived from squared structure factor amplitudes
- Used in molecular replacement and heavy atom methods for phase determination
- Calculate electron density maps using amplitudes and phases of structure factors
- Initial maps often improved through (solvent flattening, histogram matching)
Model Building and Refinement
- Fit amino acid residues into using specialized software (, )
- Refine model iteratively by adjusting atomic positions
- Improve agreement between observed and calculated structure factors
- Minimize and values
- Validate final model through multiple assessments
- Stereochemistry (bond lengths, angles)
- Ramachandran plot analysis for backbone conformations
- Evaluation of fit to electron density (real-space correlation coefficient)
Structure and Function Relationship
Structural Hierarchy Analysis
- Primary structure (amino acid sequence) determines protein folding
- Analyze secondary structure elements (α-helices, β-sheets) for contribution to overall architecture
- α-helices typically 3.6 residues per turn, stabilized by hydrogen bonds
- β-sheets formed by hydrogen bonding between adjacent strands
- Examine tertiary structure for spatial arrangement of secondary elements
- Analyze interactions stabilizing fold (, , disulfide bonds)
- Study quaternary structure to understand subunit interactions
- Implications for protein function (allosteric regulation, cooperativity)
Functional Interpretation of Structure
- Identify and characterize active sites and binding pockets
- Based on three-dimensional structure and chemical properties
- Often found in clefts or cavities on protein surface
- Infer structure-function relationships by comparing to known functional motifs
- Analyze conservation of structural features across homologous proteins
- Examples include zinc finger motifs in DNA-binding proteins, catalytic triads in enzymes
- Use molecular dynamics simulations to predict protein flexibility
- Reveal potential conformational changes relevant to function
- Timescales range from picoseconds to microseconds
- Apply computational methods for function prediction
- Docking simulations for protein-ligand interactions
- Machine learning approaches for functional annotation
Key Terms to Review (29)
Anomalous dispersion: Anomalous dispersion occurs when the refractive index of a material varies significantly with changes in wavelength, particularly in the context of X-ray diffraction. This phenomenon is particularly useful in crystallography as it enhances the contrast in scattering factors, allowing for more accurate determination of crystal structures by making certain atoms more distinguishable than others.
Batch crystallization: Batch crystallization is a process where a solute is crystallized from a solution in discrete batches rather than continuously. This method is essential in obtaining pure crystal forms of proteins for structural analysis, enabling researchers to study the three-dimensional arrangements of atoms in proteins through techniques like X-ray crystallography.
Bragg's Law: Bragg's Law is a fundamental principle in crystallography that relates the angle at which X-rays are diffracted by a crystal lattice to the distance between the crystal planes. This law, expressed mathematically as $$n\lambda = 2d\sin\theta$$, is essential for understanding how the arrangement of atoms in a crystal can be determined through diffraction techniques.
Coot: Coot is a software program used for model building and refinement in protein crystallography, particularly when analyzing electron density maps. This tool is essential for visualizing and manipulating molecular structures, allowing researchers to build models of proteins based on the experimental data obtained from X-ray crystallography. Coot facilitates the improvement of the accuracy and reliability of structural models by providing an interactive environment for refining atom positions and adjusting geometries.
Cryocrystallography: Cryocrystallography is a technique used in crystallography that involves cooling crystals to very low temperatures, typically using liquid nitrogen, to improve the quality of X-ray diffraction data obtained from them. By reducing thermal vibrations of atoms within the crystal, this method enhances the resolution of structural determination, making it particularly valuable in studying biological macromolecules such as proteins. This technique has significantly impacted both the historical development of crystallography and modern methods for determining protein structures.
Cryoprotectants: Cryoprotectants are substances that protect biological tissue from freezing damage during the cryopreservation process. They work by lowering the freezing point of water and preventing the formation of ice crystals that can disrupt cellular structures. In protein crystallography, cryoprotectants are essential for stabilizing protein crystals during X-ray diffraction experiments, ensuring that the structure can be accurately determined.
Density modification techniques: Density modification techniques are methods used in crystallography to improve the quality of electron density maps derived from X-ray diffraction data. These techniques aim to enhance the interpretability of the maps, facilitating the identification and positioning of atoms within a crystal structure. By refining the initial model or correcting errors in the density maps, these methods play a crucial role in achieving accurate and reliable structural determinations, particularly in protein crystallography.
Dialysis: Dialysis is a medical process used to remove waste and excess substances from the blood when the kidneys are unable to perform this function effectively. This technique is essential in protein crystallography, as it helps purify proteins by removing small molecules and contaminants, allowing for better structural analysis and crystallization of proteins.
Disulfide bond: A disulfide bond is a covalent linkage formed between the sulfur atoms of two cysteine residues within a protein, playing a critical role in stabilizing the three-dimensional structure of proteins. These bonds help maintain the integrity of protein conformation by providing additional stability to the folded structure, particularly in extracellular proteins that may be exposed to varying environmental conditions.
Dorothy Crowfoot Hodgkin: Dorothy Crowfoot Hodgkin was a pioneering British chemist who made significant contributions to the field of crystallography, particularly in the analysis of complex biomolecules through X-ray crystallography. She was awarded the Nobel Prize in Chemistry in 1964 for her work on penicillin and vitamin B12, which illustrated the potential of crystallography in understanding the three-dimensional structures of important biological molecules, influencing various fields such as medicine and biochemistry.
Electron density map: An electron density map is a three-dimensional representation of the spatial distribution of electrons within a crystal structure, generated from X-ray diffraction data. This map is crucial for visualizing the arrangement of atoms in a molecule, helping researchers determine the molecular structure by revealing the positions of electrons associated with the atoms.
Free-electron lasers: Free-electron lasers (FELs) are a type of laser that generates high-intensity, coherent light by using a beam of free electrons accelerated through a magnetic structure. This technology allows for the production of tunable wavelengths across the electromagnetic spectrum, making them particularly valuable in various fields, including protein crystallography and structure determination. By providing intense X-ray beams, FELs enable researchers to analyze the atomic structure of proteins and other biological macromolecules with high resolution.
Hydrophobic core: The hydrophobic core refers to the interior region of a protein that is composed mainly of nonpolar amino acids, which are repelled by water. This core plays a crucial role in stabilizing the protein's structure by driving the folding process, as the hydrophobic residues seek to minimize their exposure to the aqueous environment. The arrangement of these nonpolar residues contributes to the overall three-dimensional shape of the protein, which is essential for its function.
Isomorphous Replacement: Isomorphous replacement is a technique used in crystallography that involves replacing one atom in a crystal structure with a different atom that has a similar size and charge, allowing for the determination of the original structure through differences in scattering. This method is crucial for solving phase problems in X-ray crystallography, as it helps produce electron density maps that reveal the arrangement of atoms in the crystal. By analyzing how the diffraction pattern changes due to the replacement, researchers can infer information about the positions of the atoms in the original structure.
Max von Laue: Max von Laue was a German physicist best known for his groundbreaking work in the field of crystallography, specifically for his discovery of X-ray diffraction in crystals. This pivotal finding not only enhanced the understanding of crystal structures but also laid the foundation for the modern techniques used in both crystallography and material science today.
Molecular replacement: Molecular replacement is a method used in crystallography to determine the three-dimensional structure of a molecule by using a known structure as a reference. This technique involves solving the phase problem by fitting the model of the known structure into the electron density map derived from the diffraction data of the unknown structure. By aligning the known and unknown structures, researchers can calculate the positions of atoms in the new molecule, facilitating its detailed analysis.
Patterson Function: The Patterson function is a mathematical tool used in crystallography to determine the three-dimensional arrangement of atoms in a crystal by analyzing diffraction patterns. It simplifies the interpretation of these patterns by generating a map that shows the distances between pairs of atoms, which helps in identifying their positions without requiring phase information directly.
Phase Problem: The phase problem refers to the challenge in crystallography where the phases of the diffracted X-ray beams cannot be directly measured, complicating the process of reconstructing a crystal's electron density map. This issue is critical because the amplitude and phase of scattered waves are necessary for determining the structure of a crystal, but only the intensity (amplitude squared) is measurable. The inability to measure phase information directly leads to the need for various techniques to infer or estimate these missing values.
Phenix: Phenix is a software suite designed for the automated determination of macromolecular structures from X-ray crystallography data. It plays a crucial role in modern crystallography by streamlining various processes such as data processing, model building, and refinement, making it essential for researchers working on protein and nucleic acid structures.
R-factor: The r-factor is a measure used in crystallography to assess the quality of a crystal structure determination by comparing the observed diffraction data with the calculated data from a proposed model. A lower r-factor indicates a better fit between the observed and calculated data, reflecting the accuracy of the structural model. It plays a critical role in various fields by guiding researchers in refining models and ensuring reliable interpretations of structural information.
R-free: r-free refers to a validation metric used in protein crystallography that helps assess the quality of a model by comparing observed and calculated diffraction data. It is a critical tool for determining how well a model represents the experimental data while avoiding overfitting, as it uses a subset of reflections not included in the refinement process. This metric is crucial for ensuring that the final protein structure is reliable and accurately reflects the underlying data.
Salt bridges: Salt bridges are non-covalent interactions that occur between oppositely charged side chains of amino acids in proteins, contributing to the stabilization of protein structures. These ionic interactions play a critical role in maintaining the three-dimensional conformation of proteins, influencing their stability and function. By forming connections between different parts of a protein or between multiple protein subunits, salt bridges can help to minimize energy and increase the overall robustness of the protein structure.
Seeding techniques: Seeding techniques are methods used in protein crystallography to initiate and promote the growth of protein crystals by introducing a small seed crystal into a supersaturated protein solution. These techniques help overcome challenges associated with slow or difficult crystal growth, enabling researchers to obtain high-quality crystals suitable for X-ray diffraction analysis, which is crucial for determining protein structures.
Structure Factor: The structure factor is a mathematical expression that represents the amplitude and phase of scattered X-rays from a crystal lattice, providing crucial information about the arrangement of atoms within the crystal. It connects the real-space atomic arrangement with reciprocal space, which is essential for understanding how X-rays interact with matter, particularly in the context of diffraction patterns and their interpretation.
Synchrotron Radiation: Synchrotron radiation is a highly intense, collimated, and broad-spectrum electromagnetic radiation emitted by charged particles, such as electrons, when they are accelerated in a magnetic field. This phenomenon is pivotal in modern crystallography, as it provides powerful X-ray sources for detailed studies of material structures and biological macromolecules.
Vapor diffusion: Vapor diffusion is a widely used technique in protein crystallography that involves the gradual evaporation of a solvent, typically water, to induce crystallization of proteins. This method relies on the movement of solvent vapor into a reservoir, causing changes in the concentration of the protein solution until it reaches supersaturation, which leads to crystal formation. This process is crucial for determining the three-dimensional structures of proteins.
X-ray diffraction: X-ray diffraction is a technique used to study the structure of crystalline materials by directing X-rays at a crystal and analyzing the pattern of scattered X-rays. This method reveals critical information about atomic arrangements, symmetries, and dimensions within crystals, connecting it to various fields including material science and biology.
α-helix: An α-helix is a common structural motif in proteins, characterized by a right-handed coiled conformation where the backbone of the polypeptide chain twists into a spiral. This structure is stabilized by hydrogen bonds between the carbonyl oxygen of one amino acid and the amide hydrogen of another, typically four residues down the chain. The α-helix plays a vital role in protein folding and stability, serving as a key element in the overall three-dimensional structure of proteins.
β-sheet: A β-sheet is a common motif in the secondary structure of proteins, formed by hydrogen bonding between backbone atoms in different strands of polypeptide chains. These sheets can be parallel or antiparallel, influencing the overall stability and functionality of proteins. The arrangement of amino acids within β-sheets contributes to the protein's three-dimensional shape and plays a critical role in its biological activity.