Protein Crystallography Flashcards
Why is xray crystallography used
X ray has similar wavelength to atom
Mirrors in microscope can’t bend x rays as easily as light
What is the process of xray crystallography
Can model protein structure using electron density map
What is an atom
What is a molecule
What is a crystal
Crystals arranged in repeat units in 3D
Molecules within crystals
Nucleus small relative to electron clouds
X ray crystallography exploits interaction of x rays with electrons
How can you “ see” crystals molecular structure
What is an X ray
What is an EM wave
An EM wave is an oscillating electric (E) and magnetic (M) field
E and B oscillate at right angles to eachother and to direction of travel
What is diffraction and how can it be used to view crystals molecular structure
What is meant by oscillation *
involves repetitive motion that occurs at regular intervals
refers to any repetitive variation, typically in time, of some quantity around a central value or between two or more different states
What is the two slit experiment *
What is the volume + shape of a molecule defined as
What is an electric field
What happens when an electron is exposed to xrays
What is radians
radians are a unit of angular measurement commonly used to describe the angles involved in the diffraction pattern of X-rays as they scatter off a crystal lattice
What are sine waves
The sine wave starts at 0 when t=0, assuming no phase shift.
It oscillates between
+A and −A, producing a smooth, continuous curve.
It repeats after a period T, where T= 2 (pi)/ω
What is a cosine wave to
A cosine wave is another periodic wave, closely related to the sine wave, and is described by the equation (top of image )
Sine and cosine waves are identical but are out of phase by a quarter of a cycle
What is the phase angle of a wave
Phase (or phase angle): at a given position in space (x), the phase indicates the
position in the wave cycle (e.g. crest, trough or some intermediate part)
What are phase angles measured in
What are the properties of waves
Have can waves be added together (different types of interference )
For constructive interference:
Add the two amplitudes of the two waves to get new wave
Destructive interference:
Amplitude of new wave is zero when the two waves are added together
Intermediate interference:
Amplitude of new wave is less than the 2 amplitudes of the added waves added together
Which xrays are detected in x ray crystallography ( and why are two waves that were previously in sync before hitting 2 elections, no longer in sync)
What are the 2 ways to represent phase shift using vectors
Path difference vector
Scattering vector
How can phase shift be represented using vectors and how is path difference calculated
What is a scattering vector
Magnitude and direction of S contain information about l and the scattering angle (θ).
• At fixed l (typical in our experiments), |S| varies only with θ
• Key point: |S| is a convenient way of representing (encoding) the angle of scattering
• Note: S does not point in the direction of scattering (but we can use it to work out 2θ)
What is the general wave equation
What is the wave equation using complex numbers
What is the wave equation concerning phase shifts only
What is the wave equation when considering resultant wave
What is the equation when considering multiple electrons that all scatter waves at 2 radians
What is the equation for each scatterred wave in a molecule
What would angles of 2 radians result in
What would other angles result in
What percent of x rays are scattered by a crystal and why
How can an intensity reading be obtained for each diffracted ray
Why do you need to know the structures of proteins
How are x rays scattered by two electrons
How are x rats scattered by a single e-
What is the path difference and phase difference
What does the phase difference depend on
What is the equation for the resultant wave
How is scattering measured for more than two electrons
what is the structure factor
What is the function for electron density
What happens when an xray is applied to a molecule
What is the equation for each scattered wave from a molecule
What is the equation for the structure factor for a multi electron system
What is the equation for the structure factor for a protein
What is the equation for the structure factor for a molecule
What is the inverse Fourier transform
What are the advantages of using crystals in x ray crystallography
How are crystals organised
What can scattering from a unit cell be considered as
How does reflection from a mirror work
How does reflection from a semi transparent layer of molecules work aka layers in crystals
How do x rays go through crystals
Add all of the waves from all of the layers that have the same angle and are in phase ( so maximum amplitude signal )
What is braggs law
Why does destructive interference not obey braggs law
Why does destructive interference not obey braggs law
What do the dark spots on a x ray diffraction image mean
Why are the signals more distinguishable when crystals are used compared to a single molecule for x ray crystallography
Why is there first order diffraction for x ray crystallography*
First-Order Diffraction: For first-order diffraction,
n=1. This means that the path difference between X-rays scattered by adjacent planes of atoms must equal one wavelength. This leads to constructive interference and results in the first-order diffraction pattern observed.
Intensity and Structure Factor: The intensity of the diffracted X-rays is also influenced by the arrangement of atoms in the unit cell of the crystal, described by the structure factor. The first-order peaks are typically the most intense and informative for determining the crystal structure.
How are all possible rotations recorded for x ray crystallography
What is the first step for growing protein crystals
Compare natural vs recombinant sources of proteins
What are the pros and cons of using e. Coli in recombinant technology
Proteases used to cleave tag
How can proteins be made more crystallisable
What is the second step for growing crystsls
Name the techniques used to grow crystals
Vapour diffusion- sitting drop
Microbridge
Hanging drop
Microdialysis
Microfluidics
Liquid cubic phase plate crystallisation
Explain the sitting drop method of growing protein crystals
Explain the microbridge method of growing protein crystals
Explain the hanging drop method of growing protein crystals
Explain the LCP plate method of growing protein crystals
Explain the microfluidics method of growing protein crystals
Explain the microdialysis method of growing protein crystals
Explain the theory of crystallisation
What possible results could you get from vapour diffusion
Crystal formation
Micro crystal formation
No crystals formed
When would crystal formation occur
when a protein solution becomes supersaturated, meaning there is more protein in solution than can remain dissolved at equilibrium. This supersaturated state promotes the aggregation of protein molecules in an ordered, crystal-like arrangement
requires a stable temperature to maintain supersaturation without inducing too rapid aggregation, which could lead to precipitation rather than orderly crystal growth
When would micro crystal formation occur
When would no crystal formation occur
How can crystal formation be optimised
What are the properties of crystals
How can a model for the protein structure be built
Why is high resolution data important
These are the measured diffraction intensities (or amplitudes) obtained from the X-ray diffraction experiment.
Parameters include atomic positions, thermal factors (B-factors), occupancy factors, and sometimes more complex parameters like disorder models.
Why is knowledge about ideal stereochemistry important for building models on protein structure
What are the different ways to refine a protein structure model
Positional refinement
Temperature factor (B factor) refinement
Adding bound water molecules
What is positional refinement (in refinement of protein structure models)
What is the B factor
What is temperature factor (B factor) refinement (in refinement of protein structure models)
Why is bound water molecules added to refine protein structure models
How is refinement of protein structure models done
What should each refinement macro cycle consider
What percentage of the human genome codes for membrane proteins
Why are membrane proteins important
Give some examples of membrane protein related diseases
How are membrane proteins removed from the membrane
What are the different types of detergent
What are the 3 ways to crystallise membrane proteins
Protein-detergent complex
Addition of lipid extract
Liquid cubic phase
Explain the protein-detergent complex method of membrane protein crystallisation
Explain the addition of lipid extract method of membrane protein crystallisation
Explain the lipidic cubic phase method of membrane protein crystallisation
What are three different ways of protein engineering (for unstable proteins )
Stabilisation by random mutagenesis
Scaffolds
Fusion proteins
Explain the scaffolds method of protein engineering for unstable proteins
Eg can be done using antibodies or nanobodles
How can antibodies be used to isolate protein of interest (an example of scaffolds)
What are nano bodies
How can fusion proteins be used in protein engineering for unstable proteins
How is x ray diffraction of the protein crystals done
What data is usually collected for membrane protein diffraction
What is isotropic and anisotropic diffraction
What is the new crystallography technique
How can antibodies be used as scaffolds to increase the number of crystal contacts
