X-ray crystallography Flashcards
What does the position of a diffraction spot indicate?
level of detail/resolution
What does the amplitude of a diffraction spot indicate?
the types of atoms
What is the phase problem?
diffraction spots miss information about the relative positions of the atoms
-use molecular replacement (using similar, known structure as a model) or isomorphus replacement (add heavy metal atoms and compare diffraction patterns, heavy atoms easy reference points) to solve phase problem
How is the phase problem solved?
molecular replacement
-similar, known structure used as a model
-orientations and positions optimised to get good match between calculated and expected diffraction patterns
isomorphus replacement
-add heavy metal atoms to protein
-compare diffraction patterns
-heavy metal atoms act as easy reference points to determine the phase
How are protein crystals for x-ray crystallography obtained?
vapour diffusion
-conditions brought to a point where the protein is supersaturated and forms an ordered crystal
What makes membrane proteins difficult to study using x-ray crystallography?
-bilayer must be disrupted
-proteins aggregate in water
-detergent required
What are detergents?
amphiphilic water soluble surfactants
-polar head group, non-polar hydrophobic chain
-like lipids but more soluble
eg. alkyl maltopyranoside family of detergents popular for membrane protein studies
What is the critical micellular concentration (CMC)?
the concentration where the substances (detergent, membrane lipids and proteins) self-associate into micelles
-detergent concentration needs to be above CMC
How can the contact protein crystals have with detergent be increased (for small proteins)?
-fusing protein to larger protein domain
-partnering protein with small hydrophilic proteins (eg. Fab or Fc antibody fragments)
Structure of protein crystals
repeating lattice
-network of weak intermolecular interactions
-large channels/spaces in lattice -> get filled by water/buffer
How can the scattering pattern be analysed?
Using Bragg’s law, in which the reflected waves are interfered by a whole number of wavelengths