X-ray crystallography is used to determine the atomic structure of crystals like proteins and nucleic acids. It works by firing X-rays at a crystal sample and analyzing the diffraction pattern. This pattern is unique to each molecule and can reveal its 3D structure. To perform X-ray crystallography, the molecule of interest must first be crystallized to fix it in place. The diffracted X-ray pattern is detected and the intensity and angles are used to reconstruct the electron density and infer the molecular structure. This structure can then be deposited in databases like the Protein Data Bank for comparison with other known structures.

1. X – RAY CRYSTALLOGRAPHY
structural bioinformatics
presented by : Zirwa sarwar
presented to : dr afsha kaleem

2. X – RAY CRYSTALLOGRAPHY
• X-ray crystallography is a tool
used for determining the atomic
and molecular structure of a
crystal e.g., chemical compound,
macromolecule of life like
proteins, nucleic acids etc.
• The underlying principle is based
on X-ray diffraction pattern.

3. Why We Use X – RAY CRYSTALLOGRAPHY
• In X-ray crystallography we are dealing with the molecular structures
of the crystals that can’t be determined by the normal beam of light.
• All the molecules are converted into the crystals because the
molecular structures are good visualized in the crystal form.
• As in the normal state, a molecular continuously changes its bonding
position and it hinders in the process of structure identification of a
molecule.
• So, to hold the molecule freeze and fix at a point is achieved by
freeing it or to make the crystal of it which fix the molecule at a point.
So, the identification of target molecule structure become easier
(Ahmed and Brooks, 2022).

4. How the x-ray crystallography determine
the structure of a molecule
• When the beam of X-rays are exposed to interact with the crystal of a
molecule, the crystal will diffract or change the angle of X-rays.
• Depending on the type of molecule, e.g., protein molecule, the pattern
of diffraction varies and this diffraction pattern is unique for different
types of molecules.
• In case of a complex molecule, different varieties of diffraction occurs.
• In case of a simple molecule, when most of the region of a molecule is
blank, then the less diffraction occurs.

5. Construction of X – RAY
CRYSTALLOGRAPHY

6. 3D protein of structure
THE THREE-DIMENSIONAL
STRUCTURE OF A PROTEIN
IS DETERMINED BY X-RAY
CRYSTALLOGRAPHY

7. How the x-ray crystallography determine the structure of a
protein
• When the beam of X-rays are exposed to interact with the
crystal of a protein molecule, the crystal will diffract the X-
rays at different angles.
• The diffracted light makes a spot on the detector that is
placed on the other end.
• The detector is known as X-ray film where the diffracted
X-ray patterns can be visualized (Chojnowski et al., 2022).

8. Cont.…
• The diffraction is calculated from the distance of the center of
the axis
• From the angle of diffraction
• By the intensity of the spot
• The intensity of spot is directly proportional to the amount of
electron present in that plane.
• If the diffraction occurs away from the center point, it means the
plane of molecule contains a very high density of electrons. It
means there is a higher chance of identifying the molecule, or it
also means that most of the protein components are present in
that plane.

9. Cont.….
• The diffraction occurs close to the center point, it means the plane of
molecule contains a very low density of electrons. It means there is a less
chance of finding the fragment of the protein present in that plane.
• The density of the electrons corresponds to the intensity level.
• The angle of diffraction is different for different planes of a protein
molecule.
• The angle of diffraction tells which area of region we get the spot for and
the intensity would be helpful to identify the amount of electron density
corresponding with that area of the unit cell of protein.

10. Diffraction pattern
• When there is a diffraction going on from the nearby area of the election, that
diffraction could be in two different waves that are in the same phase, or they are
from the consecutive area of the molecule. In this case both waves are in phase are
known as constructive interference and it is going to amplify the whole
wavelength and gives a brighter spot.
• When the waves are coming from two different points of a molecule, then they are
out of phase and cancel each other and forms destructive interference and a faint
band is formed.
• The bands of light formation occurs due to the scattering of the waves (Ma et
al., 2018).

11. How to get the exact protein structure
• The electron density correlates with the appropriate structure of the protein.
Because the protein structure is composed of its secondary structure that is alpha
helix and beta sheets and their could be beta turns and loops.
• Then it can be figure out that which kind of electron density map would
correspond with the alpha helix and which kind of electron density map would
corresponding with the beta sheets.
• This corresponding idea gives the exact location of alpha helix and beta sheets
that determines the overall view about the protein

13. Protein data bank
• With the help of protein data bank, we can compare the structure of same type
of protein with each other in order to find the exactly where are the changes
that we fill the gap from the idea and information's from that database.
• So, after the determination of structure, the identification of function of protein
becomes easier.

14. references
• Ahmed, A. M. and Brooks, C. L. 2022. X-ray Crystal Structure Analysis of
VHH–Protein Antigen Complexes, ed. Springer, pp. 513-530.
• Chojnowski, G.,Simpkin, A. J.,Leonardo, D. A.,Seifert-Davila, W.,Vivas-
Ruiz, D. E.,Keegan, R. M. and Rigden, D. J. 2022. findMySequence: a
neural-network-based approach for identification of unknown proteins in
X-ray crystallography and cryo-EM. IUCrJ, 9(1).
• Ma, T.,Kapustin, E. A.,Yin, S. X.,Liang, L.,Zhou, Z.,Niu, J.,Li, L.-
H.,Wang, Y.,Su, J. and Li, J. 2018. Single-crystal x-ray diffraction
structures of covalent organic frameworks. Science, 361(6397): 48-52.