LT2 X-ray Crystallography & NMR Backbone assignment Flashcards
What type of crystal does X-ray crystallography need?
REgular, repeating array of many identical molecules (crystals) to direct an X-ray beam at
How does X-ray crystallography produce a signal?
X-ray beam onto crystal
Crystal arranged as huge number of molecules in same orientation
Scattered waves can be added up in phase and raise the signal to a measurable level
Describe crystal structure
Regular, repeating array or identical units = in each unit you may only find one protein molecule
Crystal is not a solid = contains large holes/channels filled with disordered solvent molecules
What factors need to be controlled when growing a crystal?
Can take months
Dependent on:
pH, temperature
Protein concentration
Nature of solvent and precipitant
Added ions or ligands
Gravity
By what method can you grow crystals?
Must purify the proteins first using VAPOR DIFFUSION
How do you carry out vapour diffusion?
Seal a droplet of protein solution in a chamber with a large volume of solution containing precipitant
Name two vapour diffusion methods?
Hanging drop method
Sitting drop method
Describe how the hanging drop and sitting drop method differ
The drop hangs from a siliconized glass cover over a reservoir with precipitant
Slide with depressions (seats) sealed in plastic box over a reservoir of precipitant
As the precipitant vapor diffuses into the drop, it creates a concentration gradient that encourages the protein to crystallize
Why use X-rays?
EM radiation used to visualize objects = requires the radiation to have a wavelength comparable to the smallest feature that you wish to resolve
X-ray have wavelength similar to distance between bonded C atoms ~ 1.54 angstroms
What is the function of a synchrotron?
Accelerates the electrons
What occurs when the X-rays hit the crystal?
When primary beam stroke crystal = most travel straight through it
Some are dispersed when they hit the electrons and cause the electrons to oscillate and emit X-ray in almost all direction = scattering
The X-rays emitted = interfere with each other in either destructive, constructive or anything in between manner
Define constructive vs destructive interference and how it looks when imaged
Destructive = diffracted X-rays cancel each other out
Constrictive = diffracted X-rays added together to produce diffracted beams that can be recorded as pattern
Recording can produce shades of dots anywhere in between no dot to black dot
What is the equation for Bragg’s Law?
nxwavelenth = 2d x sinθ
d = thickness of crystal
θ = incidence angle same as reflected
The condition for the two waves to stay in phase after both are reflected is that the path length CBD be a whole number (n) of wavelengths (λ), or nλ
What 3 properties define each diffracted beam?
Intensity
Phase
Position (tan2θ = r/A)
Define intensity of diffracted beam
Intensity of each diffracted spot = proportional to Amp squared (peak height) of diffracted wave
How are relative phase angles measured?
No practical way of measuring relative phase angles for the different diffracted spots
Relative phase angle = 0-360 degrees
How are electron density maps computed and what information is needed to calculate them?
Both amplitude and phase of diffracted
To compute electron density maps from diffraction pattern using inverse Fourier Transform
What does MIR stand for?
Multiple isomorphous replacement
When are heavy metal derivatives used and what for?
To obtain diffraction data of native protein crystal and at least 2 or more ISOMORPHOUS crystals
Overall phase obtained from diffraction patterns of heavy atoms
How is obtaining diffraction data from isomorphous crystal done with heavy metal derivatives?***
Usually done by diffusing different heavy metal complexes into the channels of crystal
With isomorphous crystal, the correct phase could be out of 2 possibilities
Can figure out diffraction pattern and phase with heavy metal, so only have one possibility
By comparing the two sets of data, you can determine where the heavy metal atoms are located in the crystal. These positions act as “anchors” to help you solve the phase problem.
The positions of the heavy metal atoms in the crystal are now known, and you can use this information to calculate the phases of the native diffraction data. The phases of the diffracted waves are critical for determining the actual arrangement of atoms in 3D space.
What is MAD and what does it stand for?
Multiple-wavelength anomalous dispersion
It is a powerful technique used in X-ray crystallography to determine the 3D structures of biomolecules, particularly proteins.
It exploits the anomalous scattering of X-rays by specific atoms in the crystal, typically heavy atoms or specially chosen elements like selenium.
How does anomalous scattering occur?
Heavy atom is anomalous scatterer containing protein crystal at several different wavelengths = only possible if in synchrotron
When frequency of oscillation is very similar to natural frequency of oscillation
Results in small shift in both amplitude and phase of the induced oscillation of electron
What does the strength of anomalous scattering effect depend on?
Wavelength of X-ray
Give an example of how anomalous signals are induced?
Introduce selenomethionine in place of methionine
Selenium atom replaces the S of methionine
Have strong anomalous signal a lambda that can be obtained from synchrotron
What does molecular replacement of selenomethionine in place of Met require?
HOMOLOGOUS structure
