Wolberger_Xraycrystallography

Question 1

why use x-rays to image?

Answer 1

just like light, they’re electromagnetic radiation - just with a wavelength ~1A as opposed to 1000s of A for visible

Question 2

why can’t you build an xray microscope?

Answer 2

issues focusing them, no xray lens exists, and scattering from a single macromolecule would be too weak to detect

Question 3

why crystals?

Answer 3

contains millions -billions copies of molecule of interest, gives stronger signal and enables xray scatter detection.

Question 4

unit cell

Answer 4

the fundamental building block of a crystal

Question 5

T/F. unit cells can only contain one single protein

Answer 5

False, they have have multiple

Question 6

asymmetric unit

Answer 6

describes the symmetry of a unit cell, coordinates you download from PDB are of a single asymmetric unit

Question 7

how do you grow crystals?

Answer 7

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

Question 8

T/F. Protein crystals are dehydrated.

Answer 8

False, they must remain hydrated to properly fold, typically 35-70-% solvent

Question 9

crystal contacts

Answer 9

where neighboring proteins touch each other

Question 10

why are crystals frozen before xray?

Answer 10

limits radiation damage, as xrays generate free radicals that can mess up your proteins

Question 11

synchrotron

Answer 11

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

Question 12

how can you mathematically describe the scattering of light/xrays by an object?

Answer 12

fourier transform, and you can get that object back by applying an inverse fourier transform of the scattered light

Question 13

T/F. Each electron in a molecule scatters xrays in all direction

Answer 13

True

Question 14

T/F. Electrons are electromagnetic waves, with a particular wavelength and amplitude

Answer 14

True

Question 15

What happens when multiple scattered waves collide?

Answer 15

constructive or destructive interference, depending on phase. the positions of the atoms determine phase

Question 16

T/F. Constructive/destructive interference pattern is constant regardless of the angle you view the scattering.

Answer 16

False, it is dependent upon viewing angle, which we can manipulate to gain molecular insight

Question 17

what are three properties we use to characterize waves

Answer 17

amplitude, phase, and wavelength

Question 18

why are we only looking at electron scattering?

Answer 18

because electrons are 2000X lighter than protons, they will oscillate with much greater amplitude and will account for nearly all the diffraction

Question 19

what does each region of the xray diffraction pattern represent?

Answer 19

summation of waves scattered by thousands of atoms in the protein

Question 20

what is actually recorded from the diffraction pattern?

Answer 20

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

Question 21

two cons to xray crystalliography

Answer 21

must be able to actually get crystals to grow, and hard to crystallize large or flexible assemblies

Question 22

two pros to xray crystallography

Answer 22

method of choice for highest accuracy, drives weak interactions

Question 23

what are three ways to solve the phase problem?

Answer 23

heavy atom derivatives, SAD/MAD, or molecular replacement

Question 24

heavy atom derivatives

Answer 24

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

Question 25

SAD/MAD

Answer 25

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

Question 26

molecular replacement

Answer 26

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

Question 27

how do you calculate an electron density map?

Answer 27

combination of automated chain tracing and hand fitting individual residues. easier to do the higher resolution you have

Question 28

T/F. You can tell the resolution of your crystal by how far out the diffracted dots go in your crystallograph

Answer 28

True, this is standardized

Question 29

Why don’t crystals diffract to infinite resolution?

Answer 29

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

Question 30

R factor

Answer 30

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

Question 31

What are three types of errors that can mess up your R factor?

Answer 31

poor initial phases, poor map interpretation, or model bias

Question 32

crystallographic refinement

Answer 32

a computational process in which you move atoms in the model slightly to make the R factor lower

Question 33

Rfree

Answer 33

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

Question 34

SAXS

Answer 34

small angle xray scattering, scattering from xrays in solution with macromolecule of interest

Question 35

what are the benefits of SAXS?

Answer 35

don’t need a crystal, cheap and fast, can study changes in solution

Question 36

radius of gyration

Answer 36

the effective radius of a protein, the root mean square distance of all atoms from the center of mass