Tertiary Structure Flashcards
What are the experimental approaches used to determine protein structure?
- X-Ray crystallography
- Nuclear Magnetic Resonance
What is X-Ray Crystallography?
A method used to study the structure of molecules by analyzing the scattering of X-rays by the electrons in a crystal.
What does X-ray imaging reveal in X-Ray Crystallography?
Electron density
What is the importance of the resolution of the X-ray experiment in X-Ray Crystallography?
- The sharpness of features in the electron density
- our certainty about the positions of atoms
depends on the resolution
Why are crystals required in X-Ray Crystallography?
The intensity of x-ray scattering by a single molecule is unimaginably weak,
Crystals of biological samples are required to align ~1015 molecules so they scatter x-rays in phase (constructive interference).
What limits the resolution of the diffraction measurements in X-Ray Crystallography?
Imperfections (disorder) in crystals of biological samples
Electron Density
The time-averaged distibution of electrons in a molecule
What are the steps involved in protein expression and purification?
- Clone - gene encoding protein
- Grow cells
- Lyse cells
- Separate - centrifugation for CFS
- Purification - affinity chromotography
- Check purity - SDS gel
How does protein solubility depend on concentration?
At high concentrations, protein-protein interactions can lead to the formation of aggregates, reducing solubility.
What factors affect protein solubility?
- pH
- Salts
- Polar Solvents
- Temperature
How does pH affect protein solubility?
As pH changes, certain groups go from:
neutral to charged
or
charged to neutral
This alters surface charges and interactions with water
How do Salts affect protein solubility?
Salting in - increases protein solubility
Salting out - decreases protein solubility
Affects protein surface charges & interact with water.
How do Polar solvents affect protein solubility?
precipitating agents
eg. polyetheleneglycol (PEG) soaks up water
How does temperature affect protein solubility?
Thermodynamic factors influence solubility
What are the two methods of protein crystallization?
- The Vapour Method
- The Dialysis Method
What are the two most commonly used vapour diffusion methods for protein crystallization?
- Hanging drop method
- Single drop method
What does the droplet of protein solution contain in both the methods of protein crystallization?
Purified protein
Buffer and precipitant
being allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations
How do the hanging drop and sitting drop methods of protein crystallization work?
- The droplet initially contains an insufficient concentration of precipitant for crystallization
- As water vaporizes from the drop and transfers to the reservoir,
the precipitant concentration increases to a level optimal for crystallization. - These optimum conditions are maintained until the crystallization is complete.
What is the Dialysis method of protein crystallization?
- Involves placing a soluble protein in a “dialysis button” covered with a dialysis membrane.
- The protein solution equilibrates with buffer in which the button is placed.
- This leads to supersaturation, nucleation & growth.
What are crystallization robots and high throughput crystallization methods?
- Using robots to set up and automate large numbers of crystallization experiments simultaneously.
- Carry out each step of the crystallization procedure quickly and with a large number of replicates.
- Each experiment utilizes tiny amounts of solution (100 nl drops) and is monitored by a camera which detects crystal growth.
What can exposure to X-rays do to protein crystals?
- Causes damage by removing electrons from atoms, creating free radicals which are highly reactive.
- Over time, the crystal “dies”.
How can the lifetime of a crystal be extended?
At low temperatures
near 100 K (-173⁰C).
How should protein crystals be frozen, and what are the potential drawbacks?
- Liquid nitrogen (or liquid propane).
- Frequently damaged by freezing
- can become more mosaic (i.e. broken up into tiny nano-crystals each with slightly different orientations), which can lower the resolution.
What is a cryo-protectant?
Added before freezing to help protect protein
glycerol or PEG400
What are some characteristics of electron speed compared to X-rays?
- Most electrons orbit at a speed around 1/100th c (~2x106m/s).
- In one X-ray cycle, an electron would travel 0.01Å,
- X-rays see electrons as if they are standing still.
What is the difference between constructive and destructive interference in electron scattering?
Constructive interference:
* Two electrons oscillating in the same place
* Oscillate in phase
* Scatter twice as much.
Destructive interference:
* Two electrons separated by λ/2
* Oscillate out of phase
* Cancel each other.
How are reflection planes determined in X-ray crystallography?
The “amplitude” of scattering is measured
it is proportional to the differences of electron density in the direction of “reflection planes”.
The orientation and separation of reflection planes are determined by the directions of the incoming and scattered rays.
Why is it necessary to rotate the crystal over many degrees in X-ray crystallography?
- To sample all angles.
- About 100 X-ray diffraction images are required to provide a “data set”.
- Moving the X-rays and the detector gives a new set of planes.
- Changing the angle of reflection changes the spacing (resolution).
What is Bragg’s Law?
λ = 2d sinΘ
Explains the relationship between:
* x-ray wavelength (λ)
* angle of incidence (Θ)
* spacing between planes (d) in crystallography.
Why are short wavelengths needed in crystallography?
To resolve features corresponding to small (atomic) distances in crystallography.
What happens to the diffracted x-rays emitted from collisions with electrons?
The x-ray waves can add up in phase (constructively) or out of phase (destructively),
depending on the orientation of molecules with respect to the incoming and outgoing waves.
How are the molecules within a crystal aligned in crystallography?
So that their diffraction patterns are in phase only at discrete positions that depend on the internal dimensions and symmetry of the crystals.
What is the resulting x-ray diffraction pattern recorded on in crystallography?
A 2D detector as discrete data points known as “reflections.”
How do we consider the diffraction pattern to arise in crystallography?
From x-rays “reflecting” off of discrete planes (Bragg planes) within the crystal,
consisting of equivalent atoms of the structure aligned with the incident x-ray beam.
What do the positions and intensities of X-ray reflections depend upon in crystallography?
Crystal parameters and the wavelength of the incident x-rays,
Relative intensities contain information about the structure of the molecule.
What has led to the spectacular progress in crystallography in recent years?
Improved techniques in:
* crystal preparation (robotics)
* synchrotron x-ray crystallography
* software development
What are some proteins that are difficult to crystallize, and why?
- Some proteins do not fold properly putside their native environment eg ones part of cell membrane
- their structure is altered by interacting proteins or switcing between different states
- This prevents crystal growth of gives structures that do not represent the natural structure of the protein
What experimental approaches are used to determine the 3D structure of proteins that are difficult to crystallize?
- Nuclear Magnetic Resonance (NMR)
- Transmission electron microscopy (TEM).
Nuclear Magnetic Resonance (NMR)
Only technique capable of determining 3D structure of bio macromolecules at atomic resolution in solution
Transmission electron microscopy (TEM).
Best suited to large proteins or protein complexes
What is comparative modeling?
Used to predict the 3D structure of a new protein given the structure of at least one protein with the same fold.
Technique is based on the strong tendency for structure and function to be conserved among homologous proteins.
What are the steps involved in comparative modeling?
- Fold assignment
- Target-template alignment
- Model building
- Model evaluation.
What is AlphaFold?
An AI-based protein structure prediction tool that predicts protein structures de novo (from scratch, without using previously solved proteins as templates).
How does AlphaFold predict protein structures?
Uses Deep neural networks that are trained to predict:
1. the distances between pairs of amino acids and
2. the angles between chemical bonds that connect those amino acids.
The resulting scores are then optimized through a gradient descent resulting in highly accurate structures.
What is the accuracy of AlphaFold’s predictions?
- Around 35% of the more than 214 million predictions are deemed to be highly accurate, as good as experimentally determined structures.
- Another ~45% are considered to be accurate enough for many applications.
What is the AlphaFold Protein Structure Database?
- Launched in 2021
- Contains AlphaFold-predicted models of protein structures of nearly the full UniProt proteome of humans and 20 model organisms, amounting to over 365,000 proteins.
What is ESMFold?
- An AI-based protein structure prediction tool
- Developed by researchers at Meta
- Predicts the structures of ~600 million proteins
- From metagenomic DNA from soil, seawater, and the human microbiome
How does ESMFold predict protein structures?
- Uses a Transformer-based language model, ESM-2, which allows 3D protein structure prediction at the resolution of individual atoms.
- Trained on the aa sequences of known proteins, fills in the gaps in missing sequence.
- A second step combines this with information about the relationships between known protein structures and sequences, to generate predictions.
What is the ESM Metagenomic Atlas?
- Made up of ~617 million structures generated by ESMFold in just two weeks.
- The vast majority are novel hypothetical proteins, coming from previously uncultured organisms – primarily bacteria and viruses.
- Of the 617 million predictions, 225 million (approx. 1/3) are deemed to be high confidence predictions.
