Wolberger_Xraycrystallography Flashcards
why use x-rays to image?
just like light, they’re electromagnetic radiation - just with a wavelength ~1A as opposed to 1000s of A for visible
why can’t you build an xray microscope?
issues focusing them, no xray lens exists, and scattering from a single macromolecule would be too weak to detect
why crystals?
contains millions -billions copies of molecule of interest, gives stronger signal and enables xray scatter detection.
unit cell
the fundamental building block of a crystal
T/F. unit cells can only contain one single protein
False, they have have multiple
asymmetric unit
describes the symmetry of a unit cell, coordinates you download from PDB are of a single asymmetric unit
how do you grow crystals?
most common - hanging drop vapor diffusion. you basically pipet a tiny bit of protein and crystalization cocktail (this is the hard part) onto a glass slide, then place it upside down over a well of the same cocktail. As vapor diffuses into the well, concentrations in the drop increase, forming crystals
T/F. Protein crystals are dehydrated.
False, they must remain hydrated to properly fold, typically 35-70-% solvent
crystal contacts
where neighboring proteins touch each other
why are crystals frozen before xray?
limits radiation damage, as xrays generate free radicals that can mess up your proteins
synchrotron
particle accelerator, has electrons or positrons traveling near the speed of light in a circle, radiating xrays tangent to the circle, which are harvested for an xray source
how can you mathematically describe the scattering of light/xrays by an object?
fourier transform, and you can get that object back by applying an inverse fourier transform of the scattered light
T/F. Each electron in a molecule scatters xrays in all direction
True
T/F. Electrons are electromagnetic waves, with a particular wavelength and amplitude
True
What happens when multiple scattered waves collide?
constructive or destructive interference, depending on phase. the positions of the atoms determine phase
T/F. Constructive/destructive interference pattern is constant regardless of the angle you view the scattering.
False, it is dependent upon viewing angle, which we can manipulate to gain molecular insight
what are three properties we use to characterize waves
amplitude, phase, and wavelength
why are we only looking at electron scattering?
because electrons are 2000X lighter than protons, they will oscillate with much greater amplitude and will account for nearly all the diffraction
what does each region of the xray diffraction pattern represent?
summation of waves scattered by thousands of atoms in the protein
what is actually recorded from the diffraction pattern?
so each spot is assigned an index (h, k, l value) along with the intensity of that spot. you can’t measure phase, you have to figure it out other ways
two cons to xray crystalliography
must be able to actually get crystals to grow, and hard to crystallize large or flexible assemblies
two pros to xray crystallography
method of choice for highest accuracy, drives weak interactions
what are three ways to solve the phase problem?
heavy atom derivatives, SAD/MAD, or molecular replacement
heavy atom derivatives
where you attach 1+ heavy atoms to your crystal (mercury, silver, gold etc) by soaking crystal in solution with dissolved heavy metal- you then get a heavy atom derivative and compare its diffraction pattern to the underivatized crystal
SAD/MAD
Single or multiple wavelength anomalous dispersion, make your protein with selenium or bromine (express protein in minimal medium in the presence of selenomethionine, or bromine by soaking crystal in bromine salts
molecular replacement
use structure of similar protein in PDB (>30% identity as rule of thumb), you can use this structure to provide phases for its unknown relative. You position the orientation of the PDB structure over the unknown, and once positioned you can calculate phases with Fourier transform
how do you calculate an electron density map?
combination of automated chain tracing and hand fitting individual residues. easier to do the higher resolution you have
T/F. You can tell the resolution of your crystal by how far out the diffracted dots go in your crystallograph
True, this is standardized
Why don’t crystals diffract to infinite resolution?
High resolution diffraction needs each unit cell to be identical, but proteins have flexible regions and small diffs always will exist between individual unit cells
R factor
a measure of agreement between experimental and calculated data - the diff between observed and calculated amplifture for each spot (hkl). Lower the R factor, the better the resolution. Lower limiit around 0.15
What are three types of errors that can mess up your R factor?
poor initial phases, poor map interpretation, or model bias
crystallographic refinement
a computational process in which you move atoms in the model slightly to make the R factor lower
Rfree
reliability index, where you set aside 3-5% of your spots and calculate R for this test set - it should agree with your R value
SAXS
small angle xray scattering, scattering from xrays in solution with macromolecule of interest
what are the benefits of SAXS?
don’t need a crystal, cheap and fast, can study changes in solution
radius of gyration
the effective radius of a protein, the root mean square distance of all atoms from the center of mass
