X-ray Crystallography

Synchrotron Facilities

  • Synchrotron facilities use electron sources to generate X-rays.
  • Electrons are circulated within the synchrotrons, emitting X-rays.
  • These X-rays are used to shoot through crystals, producing diffraction patterns.
  • Diffraction patterns are used to determine protein crystal structures.

Collecting Diffraction Data

  • Crystals are placed on a goniometer and rotated to collect diffraction data.
  • Data is collected over a range of angles, often up to 360 degrees.
  • Data collection occurs at small intervals (e.g., every 1 degree or 0.5 degree).
  • This process provides a comprehensive picture of diffraction data from all angles.

Diffraction Patterns and Atomic Positions

  • Diffraction patterns are related to the positions of atoms within the crystal's molecules.
  • By analyzing the diffraction pattern, the positions of atoms can be determined.
  • The diffraction pattern leads to understanding electron density and atom positions within the protein crystal.

Electron Density and Resolution

  • Electron density refers to the level of detail provided about the positions of atoms.
  • Resolution is a key factor in electron density, influencing the clarity of atomic positions.
  • The Bragg equation describes the relationship between diffraction data and electron density.
  • The concept and equation was founded by Australian researchers, the Braggs.

Angstrom Resolution

  • A 0.5 Angstrom resolution provides high detail, allowing visualization of individual atoms.
  • Lower Angstrom values indicate higher resolution, enabling the observation of atomic positions.
  • Higher Angstrom values indicate lower resolution, causing loss of detail about atomic positions.
  • As resolution decreases, detailed atom information is gradually lost, but side chain information may still be visible.
  • By 5.8 Angstroms, much of the atom and side chain information is lost, but some backbone information remains.

From Diffraction Pattern to Electron Density

  • The diffraction pattern describes the arrangement of atoms within the crystal structure.
  • X-ray beams directed through the crystal are diffracted by the atoms.
  • Each diffraction spot relates to the position of an atom in the crystal.
  • Intensities of the spots provide information about amplitude.
  • Determining atom positions requires additional information beyond intensities, such as angles of diffracted X-ray beams and whether diffraction is productive.
  • The unknown information needed to definitively determine atom positions and electron density is known as the phase problem.

Solving the Phase Problem

  • Molecular Replacement

    • Uses a known protein structure to determine phases for a similar unknown structure.
    • Phase information from a previously determined structure is used to solve the unknown structure of a related protein.

  • Multiple Isomorphous Replacement

    • Involves introducing heavy atoms into the crystal.
    • Heavy atoms produce characteristic diffraction spots.
    • The positions of these spots can be related back to the crystal structure, allowing the determination of phase information.

  • Introducing Heavy Atoms into the Protein

    • Using metals commonly found in proteins, or introducing metals like selenomethionine.
    • These atoms diffract X-ray data in a known way (e.g., known angles and amplitudes).
    • This information is then used to determine the phase.

Deriving Electron Density

  • After solving the diffraction data, an electron density map is obtained.
  • This map represents the positions of atoms within the protein crystal.

Modeling Amino Acids

  • The next step is to model amino acids into the electron density.
  • This involves fitting the known shapes of amino acids into the density map and linking them together.
  • Knowing the amino acid sequence of the protein is crucial for this step.
  • Amino acids are bonded together with known distances and angles, following specific rules.
  • Amino acid side chains also follow certain geometries, simplifying the threading process.
  • Computational programs (e.g., COT) are used to thread amino acids into the electron density and build the protein structure.

Amino Acid Characteristics

  • There are 20 different amino acids, each with unique side chains that give rise to different shapes of electron density.
  • The shape of the electron density helps in identifying the correct amino acid at each position.
  • For example, tryptophan has characteristic "bulbs" of electron density, while glycine has no side chain.
  • Lysine has a long, thin electron density. These unique shapes assist in accurately placing amino acids.

Geometry and Validation

  • The way amino acids are bonded together and the arrangement of their side chains follow a known set of geometries.
  • Bond lengths between atoms in amino acids and their relative positions must adhere to specific distances and angles.
  • These rules ensure biological relevance and structural integrity.
  • A Ramachandran plot is used to analyze the geometry of each amino acid in the protein.

Ramachandran Plot

  • The Ramachandran plot describes the possible geometries for each amino acid.
  • Each spot on the plot represents a different amino acid and indicates whether it follows the correct geometry.
  • The plot shows all possible geometries, highlighting those that are biologically plausible.
  • Amino acids need to be clustered within known, biologically relevant geometries.

Assessing Structure Quality

  • The geometry of amino acids and their fitting into known bonding patterns indicates the quality of the protein structure.
  • In a high-quality structure, all amino acids fit within the correct bonding distances and angles.
  • Poor-quality structures have incorrect bond angles and distances, leading to deviations on the Ramachandran plot.
  • The Ramachandran plot indicates the proportion of amino acids in favored, allowed, and disallowed regions, reflecting the structure's quality.
  • A good structure should have nearly 100% of amino acids in the favored regions and 0% in the disallowed regions.

Example Ramachandran Plots

  • Two Ramachandran plots can be compared to assess the quality of protein models.
  • The proportion of amino acids in favored, allowed, and disallowed regions is used to infer the better quality model.
  • A structure with 85% in the favored region and 0% in the disallowed region is considered higher quality than one with similar favored region percentage but 1.6% in disallowed regions.

X-ray Crystallography in Research

  • X-ray crystallography is used to determine tertiary protein structures.
  • In research labs, it is applied to study bacterial proteins that cause infections and diseases.
  • Understanding these protein structures helps in developing inhibitors to block their function and create antibacterials.

Bacterial Proteins and Virulence

  • Bacteria have surface and secreted proteins that enable them to cause infections and diseases.
  • These include toxins, capsules, adhesions, and flagella.
  • Adhesions function like glue, sticking bacteria to host surfaces.
  • Flagella help bacteria swim.
  • By understanding how these proteins function, inhibitors can be developed to block them, leading to new antibacterials.

Examples of Solved Bacterial Proteins

  • Auto-transporter protein involved in biofilm formation.
  • DSPA enzyme that adds disulfide bonds to proteins.
  • Protease enzyme.

DSPA Enzyme

  • The DSPA enzyme catalyzes the formation of disulfide bonds, which are required for protein stability.
  • This enzyme adds disulfide bonds to many bacterial virulence factors.
  • Without disulfide bonds, virulence factors cannot form proper folded structures and become non-functional.
  • Inhibiting DSPA can prevent bacteria from making functional toxins and adhesions.

Crystal Structure of DSPA

  • The crystal structure of DSPA enables the determination of its active site.
  • It reveals how the protein binds to other proteins to introduce disulfide bonds.
  • The mechanism involves transferring disulfide bonds through a pair of catalytic cysteines.
  • This information is used to develop drugs that bind to the surface and block DSPA from forming disulfide bonds.

Growing and Soaking Crystals with Drugs

  • DSPA protein crystals can be grown with drugs or soaked in drug solutions.
  • Drugs bind into the catalytic site of the enzyme.
  • Determining the crystal structure of DSPA with a drug bound provides information about which drugs bind and how to improve their binding affinity.
  • This approach aids in developing better drugs using structural biology.

Auto-Transporter Protein (Antigen 43)

  • Antigen 43 is a bacterial surface protein that causes bacteria to clump together and form biofilms.
  • Biofilms make bacteria resistant to antibiotics because the antibiotics have difficulty penetrating the biofilm to kill the bacteria.
  • The goal was to understand how antigen 43 promotes biofilm formation.

Crystal Structure of Antigen 43

  • The crystal structure of antigen 43 revealed a beta helix structure.
  • The protein was found to bind to itself in the crystal structure.
  • High-resolution data allowed the identification of specific amino acids involved in this self-binding.
  • The self-association of this protein explained its function.

Mechanism of Biofilm Formation

  • Bacterial cells expressing antigen 43 on their surface stick together due to the protein's self-binding property.
  • This clumping leads to biofilm formation.

Developing an Inhibitor

  • An antibody-based inhibitor was developed to block antigen 43 from binding to itself.
  • This inhibitor prevents the bacteria from sticking together and forming biofilms.

Confirming the Mechanism

  • X-ray crystallography was used to determine the crystal structure of the antigen 43-inhibitor complex.
  • This confirmed where the inhibitor bound on antigen 43 and validated the mechanism of inhibition.