Flashcards in Lecture 8 DA Deck (29):
What is the most common experimental means for obtaining 3D structures of proteins?
How does X-ray crystallography work?
Electron clouds of proteins scatter and weakly diffract the X-rays, proportional to the density of electrons.
How are atom positions determined using X-ray crystallography?
By interpreting the diffraction using an ordered array of the molecule, such as a crystal.
When doing X-ray crystallography, how many times is the crystal hit with X-rays?
Several hundred times, done as the crystal rotates.
How are electron density maps generated from diffraction patterns?
Algorithms are used, building a 3D structure.
What is the metastable region of crystallisation?
Spontaneous nucleation theoretically shouldnt occur, but is a condition where the crystal grows very uniformly.
What happens at the labile region of crystallisation? What kind of solution is needed?
Protein more likely to interact with itself than the solvent, so crystallises. A supersaturated solution.
What method is used to grow protein crystals?
Crystallisation is controlled using vapour diffusion.
How do crystals form?
When a protein solution is slowly dehydrated under controlled conditions that favour ordered lattice growth, rather than disorderred aggregates - precipitates.
Describe the process of crystallisation by vapour diffusion (3).
-A well is used, with an inverted coverslip containing a suspended droplet of protein solution.
-The well beneath the coverslip is filled with a concentrated precipitant solution reservoir.
-Water from the protein solution droplet will diffuse downwards to the reservoir in the well, via equilibrium laws.
What is commonly used as a precipitant solution in vapour diffusion?
How can multiple wells (ie. a matrix of wells) used in vapour diffusion be used to set parameters? Why is this useful?
Can be used to set certain parameters, such as a precipitant concentration gradient horizontally, as well as, for example, a pH gradient vertically.
As only a droplet of protein solution is needed, allows one to effectively determine the best conditions for protein crystallisation at once.
How are quasicrystals structured?
Their interior is disordered, but their exterior is ordered.
Where is the barrier to nucleation?
Between the labile and metastable region.
What kind of crystals are suitable for X-ray crystallography (3)?
They need to be:
-Large single crystals
-able to rotate plane polarised light
-Have few, if any, defects
What are twin crystals? Are they suitable for analysis?
They are formed when two crystal lattices intersect.
Makes it difficult to collect x-ray data relating to a single lattice, algorithms can be used.
How do cusps/clefts occur in crystals during growth?
When the solution around a crystal is depleted locally, due to crystallising too fast, causes defects.
What are crystalline aggregates?
When multiple crystals form as clusters.
How are proteins in crystals arranged in crystals (aside from being an ordered lattice)?
Arranged to fill as much space as possible, but gaps always remain, filled by solvent.
What are single crystals mounted on for analysis?
A capillary, or nylon loop.
What is the difference between a capillary mount, vs a nylon loop?
Capillary - for diffraction at ambient temperatures.
Nylon loops - for diffraction at low temperatures.
What kind of coolant is used for nylon loops and capillary mounts?
Capillary - none
Nylon loop - liquid N2/He
When rapid cooling is necessary for analysis, what is the crystal mounted on beforehand, and what does it form after cooling?
A cryoprotectant that forms glass upon rapid cooling.
What normally happens to protein crystals during cooling? What purpose do cryoprotectants have regarding this?
Microscopic ice crystals form during rapid cooling, forming ice rings that interfere with diffraction.
Seen as diffuse rings on the diffraction pattern.
Cryoprotectants prevents these crystals from forming.
What percentage of protein crystals is water by volume?
What kind of device can be used to produce high brilliance x-rays?
How are diffraction patterns converted to a 3D structure? Why is it beneficial to know the protein sequence beforehand?
Positions between the dots are measured in A/nm.
Algorithms are used.
Protein sequence is normally known beforehand.
Residues are fit to the electron densities from the data.
Missing electron densities are often found. What can they be attributed to?
Typically water molecules.