1. Radiation damage in crystalline biological samples and its influence on the Debye – Waller factor
Karolina Krystyna Sosnowska
Warsaw University of Technology
Faculty of Materials Science and Engineering

2. Outlook X – ray diffraction Debye – Waller Factor Protein structure
Radiation Damage
Data Collection
Results
Conclusions
General outlook on
The idea of the experiment: X ray diffraction analysis on single crystalline proteins of radiation damage.

3. Motivation before exposure after exposure
Analyzing flexibility of proteins via Xray diffraction
X ray distroy protein molecules
To analyze influence on the struture and DW factor
Since we use diffraction as the investigation method, I need to mention the diffraction conditions = Bragg’s Law
Not going to describe it
What is crucial to mention – in order to get clear Bragg reflection with no side maximas, we need to have relatively large
Crystal in the range of micrometers

4. X – ray diffraction Electron Density Map Structure Factor
The result of the crystallographic experiment is a map of distribution of electron density in the molecule
And since electrons are localized tightly around nuclei – well describes the molecule
Electron density map – Fourier transform of the structure factor (gained from intensity measurement)
Structure factor – describes the scattering amplitude of the unit cell
And it is obtained indirectly while measuring the intensity BUT
Phase problem – lost phase information during measurement – we dont suffer from it – we know the structure (PDB)
So we can already start the refinement of the structure
#f# - atomic form factor – depending on the atom kind and diffraction angle
#rn# - position
#Q# – scattering factor

5. Debye – Waller factor Describes the thermal motion of the atoms
Contains information of static and dynamic disorder
Debye – Waller factor
B-factor value – measures oscillations and vibrations of an atom
DW describes the thermal motion plus have info about static and dynamic disorder
Thermal motion is smearing out the position of an atom
Atoms in side chains are expected to show more freedom of movement than the ones in the main chain
B- factor is the temperature factor
Measuring how much an atom oscillates and vibrates
<Um2> mean square displacement
Teta – Bragg reflection angle
Lambda – wave lenght

6. Radiation damage Two steps process:
Primary damage – exposure to the X–ray beam
Secondary damage – chemical reactions in the material
The radiation damage in the protein structures is believed to divide into a two stage process.
PRIMARY DAMAGE - rapid process; caused by direct interaction between the radiation beam and electrons
- results in the creation of initial radiolytic products, when a photon is absorbed.
SECONDARY DAMAGE - is the reaction of these products with the protein structure.
Because of the high presence of water molecules in the protein crystals and the ability of radiolytic products to diffuse through the crystal, the subsequent chemical reactions take place causing radiation damage:
Analyzing the protein at low temperatures, diffusion of radiolytic products through the lattice slows down or ceases.
WE STUDY BOTH!

7. Protein – Lysozyme
Protein – an amino acid chain fold into unique 3D structure.
Lysozyme – a single polypeptide chain of 129 amino acids (residues) with four pairs of di-sulphide bridges.
Proteins are amino acid chains, also referred to as residues, (show them)
that fold into unique 3D protein structures,
that is strongly connected to its enzyme function.
Protein sizes range from 40 – 50 as a lower limit to several hundred residues in multi-functional proteins
2. Lysozyme is a stable enzyme, found i.e. in egg white, or tears.
It works antibiotic by attacking the cell wall of bacteria, destroying the integrity of the cell
It is large molecule, not suitable for pharmacology as it can not travel between the cells because of its siye
Discovered by chance by Alexander Fleming – like peniciline
3. Lysozyme that we investigate was taken from the hen egg white.
It is built from a single polypeptide chain of 129 amino acids
Has four pairs of CYSTALINE that form disulfide bridges between positions (seen in the picture)
These cross-bridges force polypeptide chain to fold – affects properties

8. Protein - crystallization
The hanging drop method
Parameters:
protein purity
protein concentration pH
temperature
precipitants
The goal of crystallization is to produce a ordered crystal, lacking in contaminants and is large enough to provide a diffraction pattern within Bragg condition, when hit with X – rays.
2. One of the c. methods - The hanging drop.
A droplet containing purified protein, buffer, and PRECIPITANTS which is supposed
to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations.
Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization,
but as water vaporizes from the drop and transfers to the reservoir,
the precipitant concentration increases to a level optimal for crystallization.
Since the system is in equilibrium, these optimum conditions are maintained
until the crystallization is complete
3. PARAMETERS
Change of those, results in different crystallographic modifications of the crystals

9. Protein – Lysozyme crystals
250mm
Tetragonal modification

10. Data collection All data were obtained at EMBL beam line X13 at DORIS
Experiments at 10K and 90K
8 datasets each
same dose mode
We need to use synchrotron radiation because of the large size of the protein molecules
In order to have to have the proper resolution , we need high intensity beam
2. General layout of beam line X13
The design:
flat pre-mirror
Double silicon (111) crystal monochromator
vertical focusing mirror
CCD detector
cryosystem
fluorescence detector
The sample is placed on a single axis goniometer and illuminated by the X-ray beam that is defined by two sets of slits with ionisation chambers on both sides (IC1 and IC2).
3. At each temperature, 8 successful datasets for the same crystal
In dose mode where carried out.
Dose mode – the fixed dose (around 20h) = flux density and exposure time integration
4.Proccessing the data – integration, correction, refinement
Refinement = fitting the molecular PDB model with the data obtained
In experiment, adjusting positions, … improving agreement
With the mean least square methods

11. Results The result of the refinement is the solved structure:
Space group – P43212
unit cell parameters:
a = 78,3 Å
c = 36,9 Å
We are able o extract the molecule strcuture as well

12. Results
#I plot#
Mean B factor value of temperature and residues, going along the chain
Measure of statistic disorder – 10K
The difference 90K minus 10K: dynamic disorder – most is the static one
#II plot#
B8 – B1 = time – 20h exposure
Y axis: difference b8-b1 scaled on the standard deviation and the absolute B value
Go along each atom
Some atomic groups show increase in DW factor = destroyed RD
If no jumps = no radiation damage
Peaks = more disorder – hint for RD

13. Results
#I plot#
From the higer SD than 2,5 of the mean value – SG = di-sulphide bridges atoms
We see increase over the time – RD
#II plot#
S not involved in the bonds
#III plot#
Carboxyl group

14. Results Negative differential electron density map
Positive differential electron density map
Part of lysozyme
Electron density map shown only in the middle
The same pictures but:
2. Negative density
8 – 1
3. Positive density
8-1
What means that electrons shifted from the green area to the red area

15. Conclusions
The influence of radiation damage on the structure is seen in Debye – Waller factor at:
di – sulphide bridges
carboxyl groups
Clearly seen in electron density map
The vast influence on Debye – Waller factor on di – sulphide bridges and carboxyl groups is seen in the electron density map. In present studies it is reported , that radiation damage occur the strongest in the sulphide bridges bonds in the lysozyme chain, which was expected on the other publication’s basis. Supposing, the radiation damage has some effect on the carboxyl atoms (creation COO in the structure possible). The further studies should be carried out, in order to determine on other modifications of the lysozyme crystals exterminating the radiation damage influence on the Debye – Waller factor with the special outlook into carboxyl group reactions.