Lecture 5 and 6 - X-ray Crystallography Flashcards
What is X-ray crystallography?
Involves diffraction of X-rays by crystals
Based on diffraction patterns, the electron density of a molecule can be reconstructed
Limited information about molecular dynamics
What is vapour diffusion and what are the different types?
A drop containing a mixture of protein and precipitant is placed in a chamber of pure precipitant
Water evaporated from the drop until the osmolarity of the drop and precipitant are equal, forming crystals
Hanging drop: Drop on an inverted coverslip
Sitting drop: Drop placed on a sealed well
What are unit cells?
Unit cells are the smallest unit of protein crystals held together in a specific conformation
Principle of data collection?
The darker the spots on the diffraction pattern, the more photons. These spots are indexed, and their intensity is measured
What are the benefits of cryo-cooling?
The crystals are cooled to 100K
This reduces thermal vibration, radiation damage, and disorder
This improves resolution
What do synchrotrons do?
Synchrotrons bend electrons using magnet, making them travel through the synchrotron. As they lose energy, very bright X-rays are formed.
What is Bragg’s law?
If the path difference (d) is equal to an integer multiple of the wavelength, then 2 X-rays, A and B will arrive at the atom in the same phase, leading to constructive interference.
The shorter the wavelength, the diffracted intensity becomes more sensitive to the path difference.
What is the phase problem?
Electron density cannot be correlated to the phase of a wave
What is isomorphous replacement?
Perturb the structure and diffraction pattern
Deduce the positions of the introduced heavy atoms to infer a phase change
Includes: Multiple Isomorphous replacement (MIR), Single Isomorphous Replacement (SIR), Multiple Wavelength anomalous dispersion (MAD)
What is molecular replacement?
Estimating the initial phase based on the structure of known molecules
How were specific structures experimentally determined?
Taq Polymerase (apo form): SIR using 0.1nM ethyl mercury phosphate.
E.coli Polymerase (apo form): MAD, 3 wavelengths, Se-Met Proteins
Taq polymerase (DNA bound): MR
What principle about model bias is true?
If experimental phases are weak, model bias will be strong
