X-ray Flashcards
X-ray purpose, resolution, crystals
determining 3D structures from a crystal
waves on a body are scattered and converted into image
good for large objects but limited y
resolve distance between atoms (1A=10^-10nm)
crystals: repeating ordered array (lattice) of molecules in 3D with molecules identically oriented
electromagnetic interactions with atomic electrons
y= 0,6-2,5x10^-10m (A)
5-20x10^3eV
Discoveries by Max von Lane, William Henry Bragg and William Lawrence Bragg
Max: X-rays diffracted in crystals and form characterisctic patterns on photographic film, crystals lattice like
Henry: X-ray spectrometer (reflections from crystal)
Lawrence: relation between wave, distance between planes and angle of reflection
Both conclude the diffraction pattern produced by arrangements of atoms in a crystal
reflection
ability to measure the intensity of each spot and asign an individual index (h,k,l) along with phase information - place atoms in space (spacing of spots) and then structure of molecule
eletrcton density of atoms in repeating unit of crystal - diffraction distribution of intensities
Real Space – FT— reciprocal space
p(x,y,z): atom positions —– F(h,k,l): structure factors
fourier transform
representation of some function in terms of waves with appropriate frequency, amplitude and phase
sum of sine waves is a good approximation to original unit cell, allows measure of intensity of spots (A^2) not phase
how do we get phase information
Molecular replacement (MR): with a model of a similar sequence structure you can computationally rotate and translate, calculating diffraction you would get - Alpha Fold2
Anomalous Dispersion: atomic e- orbitals ressonate at a particular X-ray wavelength - get phases and identify metals
Refinement
electron density compared to model, how well model fits the data (R-factor) and adjust to minimise difference between Fobs and Fcalc
R factor = S (|Fobs|-|Fcalc|)/ S(|Fobs|)
- R - + agreement
diffraction data and phases <-> e- density <->atomic model
difference map (omit): finding ligands - soak in/co-crytallise ligand - diff map will show where and how binds, quality of structures
Resolution
depends on max scattering angle (2qmax) - dmin in A
How do we get protein crystals?
pure in stable conditions, simples evaporation, slowly taken out of solution
factors afecting solubility: concentration of salt or other precipitant (PEG), pH, temperature
test many different conditions
screening: systematically vary concentration of precipitant with pH, concentration, temperature
Sparse matrix screening or imcomplete factorial search: set (48/96) solutions that have been found to grow crystals for systematic optimisation around those conditions.
automated systems
How do we get crystals in beam?
pick in a thin fibre loop (10um) and cool rapidly to about 100K, formation of glass (amorphous ice)
little manipulation
X-ray data collection
Oscillated about a small angle and the difraction is recorded
intensity of each reflection is measured
unique reflections - size of crystal unit cell, symmetry, resolution
electrons boiled off a tungsten filament but instead of hitting target they are accelerated to light speed and more being forced to change direction by superconducting magnets
amount of radiation (flux) depends on electron current and energy wave, speed of e- and how tight bend is
