X-Ray Crystallogrphy for Biotechnology Sem-2

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"X-ray crystallography is a technique used to determine the atomic and molecular structure of
a crystal, by measuring how X-rays are diffracted when they pass through the crystal lattice."
In this method, the crystal causes the X-rays to diffract in specific directions, and by analyzing the
angles and intensities of these diffracted beams, a 3D model of the electron density can be
constructed. This allows scientists to determine the positions of atoms within the crystal structure
with high precision.
What is X-Ray……………….?
We can define X-Rays or X-radiation as a form of electromagnetic radiation. They are powerful
waves of electromagnetic energy. Most of them have a wavelength ranging from 0.01 to 10
nanometers’.
Who Discovered X-rays?
X-rays were discovered by Wilhelm Conrad Roentgen (also spelled Röntgen), a German
physicist, in 1895.
How Do X-Rays work?
They are produced when high-velocity electrons collide with the metal plates, thereby giving the
energy as the X-Rays and themselves absorbed by the metal plate.
• The X-Ray beam travels through the air and comes in contact with the body tissues, and
produces an image on a metal film.
• Soft tissue like organs and skin, cannot absorb the high-energy rays, and the beam passes
through them.
• Dense materials inside our bodies, like bones, absorb the radiation.
X-ray Crystallography Technique

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Much like a camera, the X-Ray film develops depending on the areas which were exposed to the
X-Rays. White areas show the denser tissues, such as bones that have absorbed the X-Rays
whereas black areas on an X-Ray represent areas where the X-Rays have passed through
soft tissues.
Properties of X-Rays
The X-Rays properties are given below:
• They have a shorter wavelength of the electromagnetic spectrum.
• Requires high voltage to produce X-Rays.
• They are used to capture the human skeleton defects.
• They travel in a straight line and do not carry an electric charge with them.
• They are capable of travelling in a vacuum.
Uses of X-rays:
• Medical imaging (Radiography): To view bones, teeth, and organs
• Security screening: At airports and checkpoints
• Material analysis: To study structures of metals and composites
• Scientific research: X-ray crystallography to determine molecular structures (e.g., DNA,
proteins)
Steps in X-ray Crystallography
1. Sample Preparation
• The target molecule (e.g., a protein or DNA) is purified to homogeneity.
2. Crystallization
🧪 Protein and DNA Crystallization
Crystallization is the process of forming a highly ordered, repeating arrangement of
molecules—a crystal—from a solution. For X-ray crystallography, proteins or DNA must first
be crystallized, as X-rays need a crystal lattice to produce a diffraction pattern.
. Basic Principle
Crystallization is achieved by slowly removing solubility conditions from a concentrated
biomolecule solution, allowing the molecules to arrange into an orderly crystal lattice.

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🧬 2. Methods of Crystallization
A. Vapor Diffusion Method (most common)
• Used For: Proteins and nucleic acids (like DNA)
• Setup: A drop of protein/DNA + precipitant solution is placed in a sealed chamber with a
reservoir containing higher concentration of precipitant.
🔁 Two types:
1. Hanging Drop Method
o A drop of the sample (protein
+ buffer + precipitant) is
placed on a cover slip and
inverted over a well with
reservoir solution.
o Vapor diffuses between the
drop and the reservoir, leading
to gradual supersaturation.
o
2. Sitting Drop Method
o Similar to hanging drop, but
the drop sits on a pedestal in
the same chamber as the
reservoir.
B. Batch Crystallization
• The protein and precipitant are mixed directly
in a sealed container.
• No vapor exchange—crystallization depends
solely on initial conditions.
C. Dialysis Method
D. Microbatch Method
4. Use of Lead Screen
The primary purpose of a lead screen is to act as a shield against the intense, direct X-ray beam
that passes through the sample without being diffracted. This beam can be very powerful and

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can damage the detector, particularly in high-throughput experiments where crystals are being
screened rapidly.
5. Goniometer
use a goniometer to measure the range of motion, an instrument that measures angle
motion at a joint
6. X-ray Diffraction
• Crystals are exposed to a beam of X-rays.
• The crystal diffracts the X-rays in specific directions.
• A detector (usually a CCD or CMOS camera) records the diffraction pattern.
• X-ray diffraction is a generic term for phenomena associated with changes in the direction
of X-ray beams due to interactions with the electrons around atoms. It occurs due to elastic
scattering, when there is no change in the energy of the waves. The resulting map of the
directions of the X-rays far from the sample is called a diffraction pattern.
• X-ray diffraction to determine the arrangement of atoms in materials, and also has other
components such as ways to map from experimental diffraction measurements to the
positions of atoms.

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7. Data Collection
• Thousands of diffraction images are collected by rotating the crystal.
• These images show spots that represent reflections from planes in the crystal lattice.
• If the Spots are large and dark high show electron density.
• If the Spots are Small and light low show electron density.
8. Data Processing
• Mathematical techniques like Fourier Transform are used to convert diffraction data
into a 3D electron density map.
• Shows and calculate the Number and position of electron on crystal surface.
9. Model Building
• A molecular model is built by fitting known atomic structures into the electron density
map using software (e.g., COOT, Phenix).
10. Structure Refinement
• The model is adjusted to best fit the experimental data.
• R-factor and R-free values are calculated to assess accuracy.
Applications of X-ray Crystallography
Application Description
Structural Biology Determines the 3D structure of biomolecules like proteins and nucleic acids

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Application Description
Drug Discovery Identifies active sites for drug binding
Enzyme Mechanism Study Reveals catalytic residues in enzyme active sites
Molecular Modeling Helps in designing inhibitors or synthetic molecules
DNA-protein Interaction Studies how proteins bind to DNA
📊 Advantages
• Provides high-resolution atomic detail (as precise as 1 Ångström).
• Applicable to a wide variety of molecules.
• Well-established and reliable technique.
⚠️ Limitations
Limitation Explanation
Requires Crystals Not all proteins/DNA can form crystals easily
Time-consuming Crystallization and data analysis may take weeks/months
Radiation Damage X-rays can damage sensitive crystals
Static View Only shows molecules in one conformational state (not dynamics)

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Fou
Fourier Transform
Model Building & Structure Refinement