X ray crystellography

X-ray Crystallography

By: Dr. Ashish C PatelAssistant ProfessorVet College, AAU, Anand

• What is X-Ray Crystallography?– A form of very high resolution microscopy.– Enables us to visualize protein structures at the atomic level – Enhances the understanding of protein function.

• What is the principle behind X-Ray Crystallography?– It is based on the fact that X-rays are diffracted by crystals.

• X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a three dimensional picture of the density of electrons within the crystal.

• Because X-rays have wavelengths similar to the size of atoms, they are useful to explore within crystals.

X RAY DIFFRACTION• X-Ray Crystallography uses the uniformity of light diffraction of crystals to

determine the structure of a molecule or atom.• Then they use an X-ray beam to “hit” the crystallized molecule. The

electrons surrounding the molecule diffract as the X-rays hit them. This forms a pattern, this type of pattern is called the X-ray diffraction pattern.

Microscopy Wavelength VisualizationLight

Electron

X-Rays

300 nm

10 nm

0.1 nm or 1 Å

Individual cells and sub-cellular organelles

Cellular architecture Shapes of large protein molecules

Atomic detail of protein

Why X-Rays? Not Others?

X-ray• In 1895, Wilhelm Conrad Roentgen, a German physicist

observed, while doing some experiments with the discharge tube, that when cathode rays are allowed to fall on a metal target called anticathode placed in their path, a new kind of rays are produced. These radiations are called X-rays.

Properties of X-ray• X-Rays are EM radiations having a wavelength between 10A to 0.01A• In Free Space they travel in a straight line with a speed of 1,86,000

miles/sec (same as that of visible light)• They are Invisible to Eye, Cannot be Heard or Smelt• They cannot be Reflected, Refracted or Deflected by magnetic or

Electric Field• They show properties of Interference, Diffraction and Refraction

similar to Visible light• They Produce an Electric field at right angles to their path of

propagation• It can penetrate liquids, solids and gases.• X-rays interact with materials they penetrate and cause ionization• X-Rays also have a germicidal or bactericidal effect• X-rays are capable of producing an image on a photographic film

History• 1895: William Roentgen discovers x-rays.• 1912: Von Laue, Friedrich and Knipping publish “Interference

Effects with Roentgen Rays.”• 1914: English physicists Sir W.H. Bragg and son Sir W.L. Bragg

show that the scattering of x-rays can be represented as a "reflection” by successive planes of atoms within a crystal.

• 1915: Braggs awarded Nobel Prize

Crystals• Crystals used in X-ray crystallography are visible to the

naked eye, they contain a vast number of precisely ordered, identical molecules. A crystal that is 0.5 millimeters on each side contains around 1015 medium-sized protein molecules.

• When the crystals are fully formed, they are placed in a tiny glass tube or scooped up with a loop made of nylon, glass fiber, or other material depending on the preference of the researcher. The tube or loop is then mounted in the X-ray apparatus, directly in the path of the X-ray beam.

• The searing force of powerful X-ray beams can burn crystal through a hole, if crystal left too long in their path. To minimize radiation damage, researchers flash-freeze their crystals in liquid nitrogen.

• Crystals for x-ray diffraction must be:– Perfect - no twinning, inclusions or other imperfections– small (0.1 - 0.5 mm)

Why do we need a crystal?

• The diffraction from a single molecule would be too weak to be measurable. So we use an ordered three-dimensional array of molecules, i.e. Crystal, to magnify the signal.

• Even a small protein crystal might contain a billion molecules. • If the internal order of the crystal is poor, then the X-rays will not

be diffracted to high angles or high resolution and the data will not yield a detailed structure.

• If the crystal is well ordered, then diffraction will be measurable at high angles or high resolution and a detailed structure should result.

• The X-rays are diffracted by the electrons in the structure and consequently the result of an X-ray experiment is a 3-dimensional map showing the distribution of electrons in the structure.

X-ray crystallography

• More than 85 percent of the protein structures that are known have been determined using X-ray crystallography.

• Crystallographers aim high-powered X-rays at a tiny crystal containing trillions of identical molecules. The crystal scatters the X-rays onto an electronic detector like a disco ball spraying light across a dance floor. The electronic detector is the same type used to capture images in a digital camera.

• After each blast of X-rays, lasting from a few seconds to several hours, the researchers precisely rotate the crystal by entering its desired orientation into the computer that controls the X-ray apparatus. This enables the scientists to capture in three dimensions how the crystal scatters, or diffracts, X-rays.

• The intensity of each diffracted ray is fed into a computer, which uses a mathematical equation called a ‘Fourier transform’ to calculate the position of every atom in the crystallized molecule.

• The result—the researchers' masterpiece—is a three-dimensional digital image of the molecule. This image represents the physical and chemical properties of the substance and can be studied in intimate, atom-by-atom detail using sophisticated computer graphics software.

Steps in Structure Determination1. Protein purification.2. Protein crystallization.3. Data collection.4. Structure Solution (Phasing)5. Structure determination (Model building and refinement)

Step1:Protein Purification• Protein Purification: it is a series of processes intended to isolate one

or a few proteins from a complex mixture, usually cells, tissues or whole organisms.

• Why Protein Purification?– Characterization of the function.– Structure– Interactions of the protein.– protein are required with better than 95% purity

Step2:Protein crystallizationWhy Crystallization:

• X-ray scattering from a single unit would be weak.• A crystal arranges a huge number of molecules in the same orientation.• Scattered waves add up in phase and increase Signal to a level which

can be measured.• This is often the rate-limiting step in straightforward structure

determinations, especially for membrane proteins

• The first-and often most difficult-step is to obtain an adequate crystal of the material under study.

• The crystal should be sufficiently large (typically larger than 0.1 mm in all dimensions), pure in composition and regular in structure, with no significant internal imperfections such as cracks or twinning.

• Researchers crystallize an atom or molecule, because the precise position of each atom in a molecule can only be determined if the molecule is crystallized.

• If the molecule or atom is not in a crystallized form, the X-rays will diffract unpredictably and the data retrieved will be too difficult if not impossible to understand.

X ray infrastructure

Characteristics of crystals• The best crystals are pure, perfectly symmetrical, three-dimensional

repeating arrays of precisely packed molecules. • They can be different shapes, from perfect cubes to long needles. • Most crystals used for these studies are barely visible (less than 1

millimeter on a side).

• Proteins are difficult to crystallize because of their complexity

Methods of growing Protein crystals:Vapor Diffusion -(Hanging Drop Method):• This is probably the most common ways of crystallation. • A drop of protein solution is suspended over a reservoir containing

buffer and precipitant. • Water diffuses from the drop to the solution leaving the drop with

optimal crystal growth conditions.

Batch crystallization:• A saturated protein solution left in a sealed container to let the

crystals grow.Microbatch crystallization:• A drop of protein solution is put in inert oil and left to grow. Here

probability is of some diffusion of proteins into the oil.Macroseeding :• A crystal is grown in a highly saturated solution and placed in a

less saturated one where only growth of the crystal will occur. • Microseeding A few crystals are grown, then crushed, and put into

a final solution that combines them into a few nice crystals. This involves quite a bit of experimentation with solutions' concentrations to get the desired number of crystals.

Free interface diffusion: A container has levels of varying saturation. Crystals form initially in the highly saturated part, but as the solution mixes, it eventually only supports crystal growth.

Crystals MUST be:

Small in size:•Less than 1 millimeter

PERFECT:•No cracks•No Inclusions, such as air bubbles

Hanging Drop Method:

•1 to 5μl protein solution is suspended over a 1 ml reservoir containing precipitant solution•e.g. ammonium sulfate solution or polyethylene glycol

Exposing X‐Rays:

• The crystal is placed in an intense beam of X-rays, usually of a single wavelength (monochromatic X-rays), producing the regular pattern of reflections. As the crystal is gradually rotated, previous reflections disappear and new ones appear; the intensity of every spot is recorded at every orientation of the crystal.

• Multiple data sets may have to be collected, with each set covering slightly more than half a full rotation of the crystal and typically containing tens of thousands of reflections.

Data Collection• The source of the X-rays is often a synchrotron.• The typical size for a crystal for data collection may be 0.3 x 0.3 x 0.1

mm. • The crystals are bombarded with X-rays which are scattered from the

planes of the crystal lattice.• The scattered X-rays are captured as a diffraction pattern on a

detector such as film or an electronic device.

• In the third step, these data are combined computationally with complementary chemical information to produce and refine a model of the arrangement of atoms within the crystal.

• The final, refined model of the atomic arrangement-now called a crystal structure is usually stored in a public database.

• After the diffraction pattern is obtained, the data is then processed by a computer and the structure of the atom or molecule is deduced and visualized.

Identification • When X-rays are beamed at the crystal, electrons diffract the X-rays,

which causes a diffraction pattern. Using the mathematical Fourier transform these patterns can be converted into electron density maps.

• These maps show contour lines of electron density. Since electrons more or less surround atoms uniformly, it is possible to determine where atoms are located.

• Unfortunately since hydrogen has only one electron, it is difficult to map hydrogens.

• To get a three dimensional picture, the crystal is rotated while a computerized detector produces two dimensional electron density maps for each angle of rotation.

• The third dimension comes from comparing the rotation of the crystal with the series of images. Computer programs use this method to come up with three dimensional spatial coordinates.

• Atoms with higher atomic numbers (heavy atoms) have more electrons and therefore diffract x-rays more effectively. Diffractive power example: Fe > C > H.

• Hydrogen atoms often not located exactly due to small size and large thermal motion.

• Electron density map provides location of atoms relative to each other. – Smaller circle = higher electron density; center of circles = atom

• Bond angles, bond lengths may be determined → gives position of atoms in space.

• Connect the dots to get the molecular structure.

Electron Density Map

Molecular Structure

USES• Used to study many materials which form crystals like salts,

metals, minerals, semiconductors, as well as various inorganic, organic and biological molecules.

• Determine electron density, the mean positions of the atoms in the crystal their chemical bonds and various other information.

• Size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences among various materials, especially minerals. The method also revealed the structure and function of many biological molecules, including vitamins, drugs, proteins and nucleic acids such as DNA.

• Characterizing the atomic structure of new materials and in discerning materials that appear similar by other experiments.

• X-ray crystal structures can also account for unusual electronic or elastic properties of a material, shed light on chemical interactions and processes, or serve as the basis for designing pharmaceuticals against diseases.

In HIV:• Scientists also determined the X-ray crystallographic structure of

HIV protease, a viral enzyme critical in HIV’s life cycle, in 1989. • Pharmaceutical scientists hoped that by blocking this enzyme, they

could prevent the virus from spreading in the body. • By feeding the structural information into a computer modeling

program, they could use the model structure as a reference to determine the types of molecules that might block the enzyme.

In Dairy Science • X-ray crystallography technique has been a widely used tool for

elucidation of compounds  present in milk and other types of information obtained through structure function relationship.

• Stewart has shown that even solutions tend to assume an orderly arrangement of groups within the solution.

• Hence, liquid milk show some type of arrangement.• The mineral constituent and lactose are the only true crystalline

constituents in dairy products that can be analyzed by X-ray.

• For Analysis of Milk Stones • X-ray diffraction technique has also been applied for analysing the

chemical composition of milk stones. • Since each chemical compound gives a definite pattern on a

photographic film according to atomic arrangement, X-rays can be used for qualitative chemical analysis as well as structural analysis.

X-Ray Analysis of Milk Powder •  This technique has also been used in study of milk powder. Most

work has been confined to determine the effect of different milk  powdering processes upon structural group spacings within the milk proteins.

In Di f ferent iat ion of Sugar •  Since each crystalline compound gives a definite pattern according to

the atomic arrangement, the identification and the differentiation of the common sugars (sucrose, dextrose and lactose) is made simple by X-rays

In case of new material• X-ray crystallography is still the chief method for characterizing the

atomic structure of new materials and in discerning materials that appear similar by other experiments.

LIMITATIONS

• Must have a single, robust (stable) sample, generally between 50—250 microns in size

• Optically sample should be clear• Twinned samples can be handled with difficulty• Data collection generally requires between 24 and 72 hours

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