- Introduction of x-ray crystallography: what it s used for, how it works, applications in science - Different methods used to generate data - Case studies emphasizing the importance of the technique - Figures of what a typical 3D protein structure would look like after analysis - History of the method, how it has been developed since the first use - Advantages of this method as well as the disadvantages - Current bioanalytical research using this method to characterize unknown samples
- X-ray crystallography is a physical technique for determining the atomic and molecular structure of a crystal - This technique was first used in 1912, however for the determination of protein structures it was not used successfully until the 1950 s - This technique is still the boss method of characterizing atomic structure of new materials - First uses of this technique showed how certain atoms bond to each other. Crystallography showed the difference between aliphatic and aromatic C-C bonds - Specifically for proteins, this method is used to determine the protein s functionality, showing the conformation of the structure sites - Since the specificity of the protein s active sites and binding sites are dependent on the conformation of the protein, it is important to determine it s exact structure - Many advances in drug discovery and medical studies are due to XRC
- With the information given by a protein crystal, drugs can be designed to target a particular site of a protein - This technique is not limited to the size of the molecule, unlike NMR - Crystallographers aim high-powered X-rays at a tiny crystal, which will scatter the x-rays onto a detector - X-rays are used because they are just the right size to measure the distance between atoms in a molecule - The typical distance between atoms in a molecule is approximately 0.5 to 1.5 Angstroms, which is typically the length of the X-rays used - X-ray radiation is necessary because it is on the same wavelength as a covalent bond
- Since protein molecules are very large, their crystals diffract x- ray beams much less than crystals from smaller molecules - Proteins typically contain carbon, nitrogen and hydrogen, have fewer electrons than inorganic materials which results in less scattering of the electrons - Very strong x-ray sources are needed due to the poor x-ray scattering of proteins - Rotating anode tubes or synchrotrons are typically used as the x-ray source for this method - Detectors measure the amplitude of the diffracted x-rays on the film - Strong computer software needed to generate the 3D structures of the proteins and electron density maps - The resolution for protein structures is typically 2 Angstroms, whereas for individual atoms it is approximately 0.5 to 1.5
- XRC is an identification technique to determine the 3D structure of a protein - Similar to NMR, except not limited to size. Both of these methods are used for the analysis of protein structures - The first step of analysis is purifying and crystallizing the protein of interest - Crystallization causes the protein atoms to be oriented in a fixed way while maintaining their active conformations - To crystallize the protein of interest, the protein must be precipitated or extracted out of solution - The protein must be typically more than 99% pure to precipitate pure crystals - This is the most difficult part, due to the parameters such as temperature, ph, and concentration have to be very specific to form crystals with a pure structure
- Vapour diffusion is the most common method of initiating protein crystallization - This method consists of two sub-methods, hanging drop and sitting drop - The water from the protein droplet will vaporize and partition into the reservoir of buffer, leaving a pure protein droplet after some period of time - Crystallization can also be induced by dissolving and heating in an appropriate solvent - The main principle is that the protein needs to be supersaturated to form crystals - After the protein of interest has been crystallized, x-rays are generated and directed towards the crystallized protein
- X-rays can be generated many different ways - These generated x-rays are shot at the protein crystal structure which results in scattering, also known as X-ray Diffraction - The patterns on the x-ray film are a result of interference between the diffracted x-rays governed by Bragg s law - The blackening on the film is the result of the emulsions of the x-rays hitting the film - The crystal is then rotated so the x-rays can hit the protein from all sides and angles, and the 3D pattern will reveal the structure of the protein - The detectors however can only measure the amplitude of the diffracted x-rays, and not the phase shifts - This is why electron density maps must be created - This can be done by a method called a Fourier Transformation
- A Fourier transformation is a mathematical function, which takes the spatial arrangement of the electron density and gives out the spatial frequency (how closely the atoms are spaced) in the form of a diffraction pattern on the x-ray film - There are a few different methods used in protein crystallography to determine the phases of the structure - Molecular replacement, isomorphous replacement, multiple wavelength anomalous diffraction and singular wavelength anomalous diffraction are some of the different methods to solve the phase - Most common is molecular replacement. This method locates the orientation and position of a protein structure with its unit cell, whose protein structure is homologous to the unknown structure that needs to be determined - The obtained phases can generate electron density maps, which result in the complete 3D structure of the protein
- Study done to determine the structure of RNAP - RNAP is the central enzyme for gene expression - Proteins were purified and extracted using centrifugation - Sitting drop method was used for crystallization - A synchrotron was used as the x-ray source, and density maps were obtained using molecular replacement - Computer software for density modification Resolve was used for the analysis - The crystal structure of Archaeal RNAP had a resolution of 3.4 Angstroms. The active sites and binding sites were determined by the crystal structure Figure 1. Cellular RNAP structures from 3 different domains of life: Bacteria, Archaea, and Eukaryota. [1] Hirata, Akira; Klein, Brianna J; Katsuhiko, Murakami S. The X-ray Crystal Structure of RNA Polymerase from Archaea. Department of Biochemistry, The Pennsylvania State University, University Park, PA. Nature Publishing Group. (2008)
[2] Voss, James E; Vaney, Marie-Christine; Duquerroy, Stephane. Glycoprotein Organization of Chikungunya Virus Particles Revealed by X-ray Crystallography. Global Phasing Ltd, Sheraton House, Castle Park, Cambridge CB3 0AX, United Kingdom. MacMillan Publishers Limited. (2010) - Chikungunya virus (CHIKV) is an emerging mosquitoborne virus that cause outbreaks of human disease - Particularly invades susceptible cells from two viral glycoproteins, E1 & E2 - Glycoproteins were purified using affinity chromatography and recrystallized using the hanging drop method - X-ray source used was a synchrotron - Phases were obtained using molecular replacement. The structure used was monomeric glycoprotein E1 - The resolution of both E1 and E2 glycoproteins was 2.17 Angstroms - Refining program BUSTER Figure 2. Active sites of E1 and E2 glycoproteins that can potentially interact with CHIKV virus.
- XRC of the maltose transporter - Essentially shows how the central regulatory EIIA inhibits maltose uptake in E. Coli - Protein was purified using affinity chromatography, and crystallization was induced using vapour diffusion (sitting drop) - X-ray source was a rotating anode tube - Phase problem solved using molecular replacement, using structures of isolated EIIA - Resolution achieved was 2.2 Angstroms Figure 3. Allosteric binding of EIIA protein to the regulatory domain of the maltose transporter. [3] Chen, Shanshuang; Oldham, Michael L; Davidson, Amy L. Carbon Catabolite Repression of the Maltose Transporter Revealed by X-ray Crystallography. Department of Biological Sciences, Purdue University, West Lafayette, Indiana. MacMillan Publishers Ltd. (2013)
- Identifying molecular interactions to underlie pharmacological activity of dopamine transporters - Testing different substrates to observe activation or inhibition of dopamine release - Protein was purified using affinity chromatography and hanging drop method to induce crystallization - Synchrotron was used as the x-ray source - Molecular replacement was used to determine the phase - Phaser computer software was used to compute the 3D structure of the dopamine transporter - Resolution of the structure was 3.0 Angstroms Figure 4. Dopamine ribbon structure showing the active binding sites for potential antidepressants. [4] Penmatsa, Aravind; Wang, Kevin H.; Gouaux, Eric. X-ray Structure of Dopamine Transporter Elucidates Antidepressant Mechanism. Vollum Institute, Oregon Health & Science University, 3181 South West Sam Jackson Park Road, Portland, Oregon. MacMillan Publishers Ltd. (2013)
- Most XRC data is collected at cryogenic temperatures to minimize structural folding by the proteins - Study shows difference in structure between the different temperatures they are analyzed at - Used the signaling protein Gln-61. Purified by Affinity chromatography, crystallized by hanging drop - Synchrotron as x-ray source - Used isomorphous replacement for phase determination - Results show that cryogenic temperatures give the best resolution with the least structural conformation changes - Less radiation at room temperature which is better for maintaining the instrument Figure 5. Protein conformations shown at cryogenic temperatures relative to room temperatures. [5] Fraser, James S.; van den Bedem, Henry; Samelson, Avi J. Accessing Protein Conformational Ensembles Using Room Temperature X-ray Crystallography. California Institute of Quantitative Biosciences, University of California, San Francisco, CA. PNAS Ltd., Vol 108, No. 39. (2011)
- HIV is known as the pandemic resulting in the most worldwide deaths since the early 1980 s - Retrovirus which essentially completely dislodges the functions of the immune system - Enzyme is built of two halves, however it only has a single central active site as opposed to two terminal active sites - Thought process is to try to find a single inhibitor to block the spread of the virus within the body - Effort to find such an inhibitor is still in the works to this day Figure 6. X-ray crystal structure of HIV protease, showing the single active site between the two halves of the enzyme. [6] Kraut, Joseph. How do Enzymes Work? Study of HIV Protease. Structural Biochemistry Vol 242, pp 534. October 2008.
- Can characterize the molecular structure of any protein or enzyme - Size of the molecule does not matter - Introduces the vision of active sites of the determined molecular structure - Higher resolution produced for this method compared to electron microscopy - Very useful for the advances in current drug design and drug discovery
- Proteins must be almost completely pure - Complete analysis takes a millennial amount of time, approximately a year - The protein of interest must be present in a large enough quantity to be studied - Protein of interest can only be studied in the solid state - X-ray radiation may cause harm or destroy living tissues in biological samples - Very expensive technique - Limited areas where x-ray sources are available - Powerful software needed for analysis
- XRC is still the main method of structural determination of molecules - Widely used in the 21 st century for new drug design - Main methods used in XRC are vapour diffusion for crystallization and a synchrotron x-ray source - Phase problem can be solved many different ways: main method is molecular replacement - Major drawback is the amount of time it takes - Similar to NMR and Electron Microscopy, poses different advantages and disadvantages - To this day, 15 Nobel prizes won for discoveries using this method between chemistry and physics