This ppt discusses in detail the process of X ray Crystallography. Made by the following 3rd year Bs-Ms students of IISER Kolkata: B Sri Sindhu Rasiwala Hassan Shabbir Ritam Samanta Himanshu Gupta Sakshi Ajay Shrisath Aditya Borkar Diana Denzil Fernandez Neha Kumari .Sowmya Anjali Mohan Debanjana Mondal Aanandita Gope Shruti Santosh Sail

1. X- Ray Crystallography

2. Wilhelm Röntgen
Discovered X- Rays in 1895
Max Von Laue
Discovered of X-Ray Crystallography in 1912
Won Nobel Prize in 1914
HISTORY

3. William Henry Bragg & William Lawrence Bragg
Observed that the X-ray diffraction pattern can be used to determine the positions of atoms in the crystal
and together formulated Bragg’s Law and won the Nobel Prize in 1915.
nλ = 2dsinθ

4. Dorothy Hodgkin was awarded the 1964 Nobel Prize in
Chemistry for solving the atomic structure of molecules such
as penicillin and insulin, using X-ray crystallography.
In 1962, J.C Kendrew and M. Perutz received Nobel Prize in
Chemistry for determining the structure of soluble proteins such
as haemoglobin and myoglobin using X-ray Crystallography.

5. In 1962, Crick, Watson and Wilkins won the Nobel Prize for Franklin’s work!

6. What is X- Ray Crystallography?
● X-Ray crystallography is a scientific technique used to determine the atomic or
molecular structure of a crystal using a beam of X-rays focused on the crystal and
allow them to diffract into many directions.
● By measuring the angles and intensities of the diffracted beams, we get the 3D
picture of the electron density within the crystal.
● With the help of mathematical techniques and Fourier transform of the diffraction
pattern, the spatial distribution of atoms in the crystal lattice is revealed.

7. ● From these pictures and data gathered the chemical bonds, disorder and
other valuable information regarding the atoms can be obtained.
● In order to obtain a high-quality diffraction pattern, a well-ordered and high
purity crystal is required, which turns out to be one of the limitation of this
technique.
● With the help of goniometer a crystal is rotated to different angles to obtain
complete diffraction pattern.

8. ● This method has been proved to be very useful to know the structure and
function of many biological molecules such as DNA, Proteins, Vitamins,
Drugs, etc.
● Recent advancements in this technique it had made possible to know the
structural analysis of large complexes such as Viruses.

9. How does it work?
● The basic principle of X-Ray Crystallography is based on the principle of
diffraction.
● When X-Ray beam strikes against the crystal, the closely spaced atoms in the
crystal lattice diffracts the beam into different directions.
● By studying the angle of the diffracted rays, the 3D structure of any crystal
can be determined.
● The wavelength of X-Rays (1 A°) is comparable to the interatomic spacing in
crystalline solids.

11. ● Bragg’s law is a fundamental law used to calculate interplanar spacing used
in X-Ray diffraction spectra.
● The collected data from X-Ray diffraction is subjected to fourier transform
process.
● The electron density around the crystalline atoms gives us the spatial
distribution of atoms in the crystal lattice.

12. X ray instrumentation
● X-rays are generated from X-ray vacuum tubes consisting of an anode and a
cathode which are heated by an energized tungsten filament.
● When the tube is heated, electrons come out of the cathode and goes toward
the positive-biased anode.
● The intensity of the X-ray beam can be determined by raising the filament and
cathode temperature, which in turn increases the electron current.
● The kinetic energy of the electron can be determined by the voltage difference
between the anode and cathode which can be used to determine the
penetrating power of the emitted X-ray photon.

13. 1. X-Ray Tube
2. X-Ray Detector
3. X-Ray Sample Holder
4. Collimator
5. Monochromator
Essential components of a X-Ray Diffractometer

14. X-Ray Tube
The heart of the X-Ray Diffractometer.
The cathode, which is made of tungsten, is heated internally so that some electrons have enough
energy to escape the surface.
The power source applies a high voltage (high negative potential) between the anode metal and
the filament (ground potential).

15. X-Ray Sample Holder
X-ray sample holder is a device used to
fixate sample surfaces. It is usually an
aluminum plate with approximately 2mm
thickness and a square hole in the middle
with a film over which helps in diffracting
the beam
Collimator
A collimator is a tool that is used for
converting a point source divergent beam
of radiation to a parallel beam. It is made of
a tube with a convex lens at one end and a
variable aperture on the focal plane at the
other end.

16. Monochromator
Using single-crystal monochromators
can result in a beam of radiation with a
more constrained wavelength
dispersion. The monochromator
functions by reflecting wavelengths that,
for the specific d spacings of the
monochromator, follow Bragg's Law
X-Ray Detector
The flux, geographical distribution, spectrum,
and/or other characteristics of X-rays are
measured by X-ray detectors. The radiation's
energy is transformed into forms that can be
identified visually or electrically in X-ray
detectors. The detector material typically
absorbs the photons, and energy transfer
occurs by ionisation.

17. How Does Everything Work Together
A rotating anode (A) rotates with the aid of a
rotor (R) and its bearings, producing an X-ray
focus point around the anode target (T).
The filament circuit is shown with the cathode (C)
in green. These parts are all contained within the
evacuated tube envelope (E).
The stator (S), which drives the rotor to rotate, is
located just outside the envelope. A dielectric
cooling oil (O) surrounds the tube envelope, and
an expansion bellows (B) that is normally
attached to a regulator switch prevents the
heated oil from over-expanding.
The tube window (W), which is commonly made
of aluminium or beryllium, lets an X-ray beam out
while the remainder of the housing is made of
lead or copper to dampen stray X-rays.

18. Procedure
STEP 1 - Crystallization of proteins: The process of X ray crystallography starts by
crystallizing our target protein.
Four essential steps are performed sequentially for protein crystallization:
1. Purification of the protein: Estimate the purity of the protein in numerical
units and if not pure (usually >99%), redo the purification process.
2. Precipitation of the protein: The protein must be precipitated by dissolving
it in a proper solvent(usually we use water- buffer mixture,or organic salt such
as 2-methyl-2,4-pentanediol).
3. Supersaturating the protein solution: This step is achieved by adding a salt
to the concentrated solution of the protein.
4. Wait until the crystals grow: Since nuclei crystals are formed this will lead to
obtaining actual crystal growth.

19. STEP 2 - Production of diffraction patterns
• X-ray diffraction is based on the interaction of monochromatic X-rays and crystal
structures.
• These X-rays are produced by a cathode ray tube,filtered to produce
monochromatic radiation,and collected to focus and direct to the sample.
• The crystal is rotated by small angles to allow the X-rays penetrate the protein
crystal from all possible sides.

20. • The aperture of the selected area determines the width of the transmitted beam
and focusses the diffraction pattern of the central lens onto the screen.
• The pattern thus formed on the emulsion due to scattering unmasks valuable
information about the structure of the protein.
• The intensities and positions of the spots are thus the basic experimental data of
this crystal analysis step.

21. Obtaining
Protein
Protein
Purification
Protein
Crystallization
Data
Collection
Structure
Determination

22. STEP 3 - Analysis of Diffraction Patterns and determination of protein
structure
• The electron density map is obtained by deducing the data of measured
intensities of the diffraction pattern on the film.
• A computer based Fourier Transform operation is applied to the intensities on
the film to reconstruct the electron density distribution for the crystal.
• This mapping technology reveals a 3D representation of the electron
densities observed through the X-ray crystallography.
• While interpreting the electron density map, resolution shouldn’t be ignored.
A resolution of 5A°-10A° can help to visualize the structure of polypeptide
chains, 3A°- 4A° of groups of atoms, and 1A°-1.5A° of individual atoms.
The protein structure is determined by finding the exact orientations and
arrangements of the different types of amino acids present in the protein.

23. Advantages and Disadvantages of X-ray crystallography over other techniques
(Green = Advantage, Red = Disadvantage of the respective techniques)
Comparison of X-ray and Cryo-EM
X-ray crystallography Cryo-EM
High resolution Low resolution
Broad molecular weight range Can be applied to high molecular weight samples only
Cheaper technique Costly technique
Only crystalline structures can be structurally analysed Crystalline structures, along with other types of
compounds, can be analysed
Difficult to crystallise large complexes and membrane
proteins
Easier option for large compounds and proteins
High amount of sample required Low protein amount yields the results
Crystalline structures might not be in their native state
in solution
Biological molecules are imaged in their native state

24. Comparison of X-ray and NMR
X-ray crystallography NMR
Generates a unimodal, which is easy to visualize and
interpret
Generates a lot of structures, which makes the data
difficult to analyse
Can be applied to proteins having molecular weight
greater than 200kDa
Cannot be applied to proteins having large molecular
weight
Can work when protein is not in a high concentrated
solution
Require high concentration of soluble proteins
Only crystalline structures analysed Other structures also analysed
Only used in protein crystals, and crystal contacts and
distort the protein structure
Doesn’t require a crystal and is not affected by crystal
contacts
Studying of domain motions of proteins is not possible Domain motion studies can be done
No direct determination of secondary structures Secondary structure can be determined with very less
data
Non-dynamic/Static technique Dynamic technique

25. Comparison of X-ray and SAXS
X-ray crystallography SAXS
High resolution Low resolution
Better signal-to-noise ratio SN ratio low
Only applied to crystalline molecules Other molecules like flexible proteins can be analysed

26. APPLICATIONS OF X-ray Crystallography
● Differentiation of sugar: Since each crystalline compound gives a definite pattern according to
the atomic arrangement, the identification and the common sugars(sucrose ,dextrose and
lactose) is made simple by X-rays.
● In HIV, Scientist also determined the X- ray crystallographic structure of HIV protease, a viral
enzyme critical in HIV life cycle in 1989. 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, this technique has been widely used for elucidation of compounds present
in milk and other types of information obtained through structure function relationship.
Hence, liquid milk should, and does 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.

27. ● 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 process upon
structural group spacings within the milk proteins.
● Analysis of Milk stone, This technique has also been applied for analyzing 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.
● To measure thickness of thin film and multilayer.
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28. CASE STUDY #1

29. Structural characterization of aspartate‑semialdehyde
dehydrogenase from Pseudomonas aeruginosa and
Neisseria gonorrhoeae
● Aspartate-semialdehyde dehydrogenase (ASADH) is an enzyme involved in
the aspartate biosynthetic pathway.
● This enzyme catalyzes the reductive dephosphorylation of aspartyl phosphate
to aspartate semialdehyde.
● This pathway is essential for producing essential amino acids and metabolites
in the microorganisms, and inhibition of ASADH is fatal; hence it has proved to
be an attractive target for antimicrobial drugs.
● Mammals including human beings do not have a homolog of ASADH or require
this pathway make this an important pathway to target in microorganisms.

30. The schematic below shows the conversion of aspartyl phosphate
to aspartyl semialdehyde catalyzed by ASADH enzyme

31. ● Antimicrobial drug resistance is an urgent and ongoing medical threat.
High-resolution structures of enzymes that are critical for microbial survival
represent an essential starting point for developing new antibiotics.
● Here we present the crystallographic structures of P. aeruginosa and N.
gonorrhoeae ASADH proteins, representing ideal platforms for potential in
silico drug screening.

32. Schematic representation of the structure of ASADH from N. gonorrhoeae (6BAC)
(A) The monomeric N. gonorrhoeae ASADH, with α helices coloured cyan and β-sheets coloured purple. The N-termini
and C-termini are shown in yellow and green, respectively.
(B) Topology map of N. gonorrhoeae ASADH showing helices (cylindrical) and directional beta sheets (arrows) analysed
in PDBsum.
(C) N. gonorrhoeae ASADH dimer, with one monomer coloured as for (A) and the second monomer coloured in yellow.

33. Schematic representation of the structure of ASADH from P. aeruginosa (5BNT).
(A) P. aeruginosa ASADH structure with α helices colored pink and β-sheets colored green. The N terminus (pale yellow) and
C terminus (pale green) are depicted.
(B) Topology map of P. aeruginosa ASADH showing helices (cylindrical) and directional beta sheets (arrows) obtained from
PDBsum.
(C) P. aeruginosa ASADH formed a dimer with one NADP molecule (yellow) bound within each protomer.

34. NADP binding in P. aeruginosa (5BNT), H. influenzae (1PQU) and V. cholerae (3PZR) ASADH proteins.
Schematic of hydrogen bonds (H-bond) between bonding residues of P. aeruginosa ASADH and NADP.
NADP (yellow) bound within the P. aeruginosa ASADH dimer and active site residues within the NADP binding site cavity that are important for
H-bonding (green).
(C) Residues responsible for NADP H-bonding in P. aeruginosa (5BNT, purple), H. influenzae (1PQU, blue) and V. cholerae (3PZR, green) show several
conserved residues in NADP binding.
(D) Superimposed structures of P. aeruginosa (5BNT, purple), H. influenzae (1PQU, blue) and V. cholerae (3PZR, green) showing the binding surface
of the ASADH proteins (grey) modelled in SeeSAR Version 9.2.

35. Phylogenic tree of ASADH proteins. The fungal (blue), bacterial (purple) and archaeal (orange) ASADH
proteins and the corresponding PDB ID are shown.
*An asterisk indicates a non-pathogenic species.

36. CASE STUDY #2

37. X-ray diffraction analysis of the DNA-binding domain of
human heat-shock factor 2
Heat Shock factors
● Heat shock factors (HSFs) are a family of transcription factors that play a crucial role in
regulating the expression of genes in response to various stressors, including heat shock,
oxidative stress, and heavy metals.
● These proteins are found in a wide range of organisms, from bacteria to mammals.
● In response to stress, HSFs bind to specific DNA sequences known as heat shock elements
(HSEs) located in the promoter regions of heat shock genes.
● This binding triggers the transcription of these genes, leading to the synthesis of heat shock
proteins (HSPs), which help cells to cope with stress by preventing protein misfolding and
aggregation.

38. ● In addition to their role in stress response, HSFs are also involved in various
cellular processes, including development, differentiation, and apoptosis.
Dysregulation of HSF activity has been linked to a variety of diseases,
including cancer, neurodegeneration, and aging.
● There are several different types of HSFs, each with unique properties and
functions. For example, HSF1 is the main regulator of the heat shock response,
while HSF2 is involved in development and differentiation.

40. A complete diffraction data set with 360 rotation was collected to 1.32 A˚ resolution on beamline BL19U1 at
SSRF. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 65.66,
b = 64.42, c = 93.25 A˚, α=β=γ=90.

41. Conclusion
● The HSF-DBDs from K. lactis and D. melanogaster have had their structural
characteristics determined thus far. These HSFs are orthologous to mammalian HSF1,
however, HSF2 displayed a lower degree of sequence similarity to them.
● Growing evidence has identified HSF2-specific biochemical characteristics and
functional activities, such as its DNA-binding affinity and sumoylation patterns that
differ from HSF1's.
● However, the lack of mammalian HSF structures makes it difficult to understand the
structural underpinnings of these variations.
● The most conserved functional module in HSFs is the N-terminal DNA-binding
domain (DBD), which is also the only domain that can crystallize.