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Structural Biology Lesson 9 - Coggle Diagram
Structural Biology Lesson 9
X-Ray Diffraction (XRD)
1. Sample Preparation - Lesson 8
High concentration of protein is first obtained.
Choose a protein buffer in which the protein can maintain their stable structure.
The protein solution is then gradually brought to supersaturation where nucleation takes place in the nucleation zone.
Crystal formation occurs and is grown to the typical size of 0.3mm by 0.3mm by 0.1mm.
Confirm that the protein molecules in the crystal have retained their function.
Why is crystal used?
helps to amplify the signals, making the signal stronger as crystal will normally contain more than 1000 molecules
using only a single protein molecule may be burnt by X-rays has a short wavelength and will be too intense, using protein crystal will prevent that as there would be a lower chance of all protein molecules being destroyed.
ensures that the result are not overwhelmed by the 'noise' by other elements in XRD system unlike when using a single protein molecule.
2. Data Collection & Processing
Principles
When the X-ray wave hits and passes through 2 atoms of protein molecule in the crystal, the wave would be diffracted due to the gap between the 2 atoms, creating diffracted rays which leads to the creation spots of a diffraction pattern.
More protein molecules -> more overlapping of the same signal -> the darker the spot
The crystalline structure causes a beam of incident X-rays to diffract into many specific directions. The angles and intensities of the diffraction pattern can be used to form the electron density map. From this, the mean positions of atoms, chemical bonds and other information of the protein can be determined.
How is it done?
Remove cover slip and fish out crystal with a small nylon loop
Surface tension of the liquid in the loop holds crystal in place
Mount loop on goniostat in a stream of
nitrogen gas
Nitrogen gas
- cool down crystal to prevent it from melting or protein from being destroyed
Run the machine and collect diffraction pattern
High Quality Raw Data is important
as it will produce a high resolution electron density map (
1.2Å
)
=
more accurate 3D model to be formed
=
less refinements will be needed to produce the final structural model with
high
accuracy.
X-Ray Crystallography
Wavelength of light/radiation used has to be smaller than the distance between 2 atoms for better
resolution
.
Resolution
- the ability to separate 2 distinct entities
Wavelength used:
X-ray -> 10^-7
3. Model Building
Phasing
and
Indexing
of diffraction data is carried out
The diffraction data is converted into a 3D model of the electron density using software
Using several computational analysis, build the atomic model of the protein structure and refine the structure
4. Refinement
Comparison between real (observed) and predicted pattern / structure.
Tweaks and changes to the protein structure should be done to increase accuracy of structural model.
Verification by
Ramachandran
Once verified, protein structure can be put in data bank
Both Methods undergo 4 phases
Sample Preparation
Data Collection & Processing
Model Building
Refinement
Nuclear Magnetic Resonance (NMR)
4. Refinement
Comparison between real (observed from NMR Spectrum) and predicted pattern / structure.
Tweaks and changes to the protein structure should be done to increase accuracy of structural model.
(Compare the
1D, 2D, 3D NMR data
set to the model then refine it till the observed NMR spectrum matches the predicted one)
Verification by
Ramachandran
Once verified, protein structure can be put in data bank
1. Sample Preparation
DNA containing protein of interest is transfected into bacteria or certain mammalian cells to produce protein in large amount with NMR active isotopes.
Feed the bacteria / mammalian cells with carbon-13 and nitrogen-15 labeled glucose or amino acids
Once protein is obtained from the cells, it is purified then used for NMR by resuspending protein in NMR buffer (
D2O
)
Note: H2O is not used as it will produce a NMR spectrum line as well due to 1H having a net spin
Why carbon-13 and nitrogen-15 isotopes?
carbon-13 & nitrogen-15
have net nuclear spin as there are different numbers of protons and neutrons to create a magnetic field -> atomic nucleus becomes a magnet
e.g. carbon 13: 6 protons spin in one direction & 7 neutrons spin in another direction -> overall there is net spin
not carbon-12 & nitrogen-14
no net nuclear spin as same number of proton and neutrons which cancels out one another
e.g. carbon-12: 6 protons spin in one direction & 6 neutrons spin in other orientation -> overall no net spin
2. Data Collection & Processing
Principle
Nuclei have net spin, behave like small magnet.
If magnetic field applied, energy transfer is possible. Pulse of radio waves at resonant frequency provide energy and absorb by nuclei.
Nuclei spin at higher energy generate magnetic field opposing external magnetic field.
In absence of radio frequency, energy level drop -> energy is released at the same frequency and can be measured by receiver.
The precise resonant frequency depends on effective magnetic field at nucleus, which is affected by
=> electron shielding, which in turn depends on
=> nucleus’ chemical environment, such as neighbouring atom, bond strain
This produces chemical shift which is measured by NMR spectrometer and yield NMR spectrum used to build protein model
3. Model Building
NMR active atoms produce characteristic chemical shift depending on neighboring atoms.
Different types of NMR can be created with the different types of isotopes
1D, 2D, 3D
(low to high resolution)
2D
data from two isotopes are used
e.g. 1H signal and 15N signal
Atomic distance and bond
angle can now be calculated
1D
only data from one isotope is used
e.g. 1H
3D
data from all three isotopes are used
e.g. 1H signal, 15N signal and 13C
