X-ray Crystallography

Question 1

What are five pros of X-ray crystallography?

Answer 1

1) High resolution/ accurate structures
2) Easy for model building
3) Broad molecular weight range
4) Gives clues on dynamics, possibly on multiple states
5) Solvents are often observed - water shells will affect protein conformation

Question 2

What are five cons of X-ray crystallography?

Answer 2

1) Crystals sometimes take years to grow
2) Difficult diffraction/phasing
3) Crystal packing artifacts
4) High sample purity and homogeneity required
5) Requires lots of protein (mg)

Question 3

Define diffraction

Answer 3

The bending and spreading of waves as they pass through an aperture. It creates alternating bright and dark fringes of light.

Question 4

Define scattering

Answer 4

The redirection of waves in many different directions due to particles or irregularities in the medium. Results in uniform distribution of light.

Question 5

Define resolution

Answer 5

The shortest distance between two points on a specimen that can be distinguished by the observer.

Question 6

What are 3 advantages to using X-rays in crystallography as opposed to other light wavelengths?

Answer 6

• Small wavelength (λ0.03-3nm)
• Good penetration
• Easy to generate

Question 7

What are 2 disadvantages to using X-rays in crystallography as opposed to other light wavelengths?

Answer 7

• Hard to focus
• Damage the sample

Question 8

Why are protein crystals not as sturdy as ionic crystals? What is something they have in common?

Answer 8

Both form lattice structures, but salt/ionic crystals have very small spaces between the molecule while protein crystals have large spaces between the proteins which are filled with water and other ions.

Question 9

What is the “Witch Test”?

Answer 9

Take a crystal and try to break it.

• If it doesn’t break it is more likely a bad salt crystal.
• If it breaks easily then it was likely a good protein crystal.

Question 10

Define Crystal

Answer 10

A material whose contituents, whether they be atoms, molecules or ions, are arranged in a highly ordered microscopic structure that forms a lattice which extends in all directions (x,y,z)

Question 11

What is a pro and a con to protein crystals having solvent channels?

Answer 11

Pro: It allows them to passively diffuse/soak in drugs/small molecules and peptides to see how they may respond (may not always work)

Con: It makes them less durable

Question 12

Why is single protein scattering weak and difficult?

Answer 12

It is very hard to dilute to one protein, and would require an X-ray laser to pin point it. This would not be practical.

Question 13

What is pI (isoelectric point) and what does it tell you?

Answer 13

the isoelectic point is the pH at which the net charge of a protein molecule is zero. Thus, a protein above their pI should be negatively charges, and a protein below their pI should be positively charged.

Question 14

What factors affect how proteins grow?

Answer 14

1) pH
2) Kinetics [Precipitant], [Protein], Temperature
3) Type of precipitant and solvent

Question 15

What is a dielectic substance?

Answer 15

• a non-conducting substance that holds electrical charges. (water)
• It is better able to stabilize charges on a protein.

Question 16

What makes for better protein crystals? Homogeneous nucleation or heterogeneous nucleation?

Answer 16

homogeneous protein because they create regular repeating units.

Question 17

What are some obstacles in forming protein crystals?

Answer 17

• impurities
• aggregation
• flexible domains
• flexible tails
• cleaved proteins
• unfolding

Question 18

Should proteins be dehydrated during crystallization?

Answer 18

No, They need a hydration shell to maintain their structure.

Question 19

What are the components of a crystallization mix and what do they do?

Answer 19

1) Precipitant helpers (Salts and Polyethylene glycol (PEG)). They decrease solubility

2) Additives. stabilize protein/crystal contact

3) pH Buffer. Alter charge distribution

Question 20

What are some advantages of the vapor diffusion method of protein isolation?

Answer 20

• Small volumes required.
• vapor diffuses gradually over time and controlled with temperature, so dehydration can be avoided.
• some changes can be made during the process by opening up the slide and adding something new.
• easy to pick up crystals with a loop.

Question 21

What is one advantage and one disadvantage sitting drop vapor diffusion has over hanging drop?

Answer 21

• Less likely to be knocked off so better for high-throughput situations
• Crystals can get stuck at the bottom where they are hard to retrieve without breaking the crystal.

Question 22

Why is pI not used to determine the pH to best crystalize a protein?

Answer 22

It simply does not match up with the observed reality of where a protein will most frequently crystalize.

Question 23

Describe a sparse matrix screening

Answer 23

A trial and error procedure where multiple different conditions are tested to determine which is best for protein crystallization.
(2 [protein]) x (2 temp) x (5pH) x (5 precip) x (5 [precip]) etc….

Question 24

What are three types of crystallization screens which can help us tune the conditions of crystallization?

Answer 24

• Fine grid search (fine pH or salt gradient)
• Factorial design
• Sparse matrix

Question 25

What are some advantages and disadvantages of dialysis crystallization ?

Answer 25

Advantages:

• sometimes yield better crystals
• Have the option of salting in for proteins that like high salt

Disadvantages:

• low throughput
• need lots of protein
• usually results in more lose of protein.

Question 26

What are three methods of distinguishing between a salt and protein crystal?

Answer 26

• Witch / crush test (If easily crushed then a protein)
• Dyes (If changes color to dye then it is a protein with channels)
• Fluorescence of the aromatic amino acids tyrosine and tryptophan under UV radiation. Does not work if no aromatic a.a. in a protein

Question 27

What is the Nobel prize winning trick to get proteins to crystalize?

Answer 27

By replacing the flexible loops with a lysozyme the protein was able to be crystalized.

Question 28

What are some “tricks” to getting protein crystallization?

Answer 28

• Cross-linking - terminal amino groups of lysines will bind to other lysines which helps solidify it. While this can cause denaturation, it is good in hanging drop salting out experiments which occur over a long period of time.
• Lysine methylation - reduces charge on lysine residue. Will require a second purification
• Addition of ligand

-Reducing agent - prevents cysteine oxidation which creates radicals and sulfenic acids.

• Improve cryo - freeze crystals in liquid nitrogen to reduce damage from X-rays.

Question 29

How can a non-crystalizing protein be rescued?

Answer 29

using stabilizing molecules

Question 30

How can fluorophores be used to determine the extent of crystallization?

Answer 30

Fluorophores will be quenched when the protein is more tightly folded so that it creates a hydrophobic pocket for the fluorophore to go.

Question 31

Where in an X-ray crystallography image is the resolution highest?

Answer 31

The further away from the center it is.

Question 32

What is a sign of a protein in good cryo?

Answer 32

it forms a transparent glass (good) as opposed to an opaque crystal (bad).

Question 33

What is the relative intensity of spots in a Fourier Transformation proportional to?

Answer 33

The density of the material in the unit cell

Question 34

What is the relationship between number of electrons an atom has and its scattering?

Answer 34

More electrons means a higher scattering.

Question 35

What are the steps to an X-ray crystallography experiment?

Answer 35

1. Produce and purify protein
2. Grow protein crystal
3. Expose crystal to X-rays and measure the diffraction pattern
4. Get phases
5. Calculate electron density maps
6. Build model
7. Refine model

Question 36

What is more important in getting the image, amplitude or phase of a protein?

Answer 36

The phase

Question 37

What is molecular replacement in the context of X-ray crystallography?

Answer 37

It is a computational method which estimates of phases from similar proteins (>50% similarity) to calculate the electron density maps of a protein which can be used to make a model which we can then get a new phase from.

Question 38

When can the phase of one protein be used in molecular replacement?

Answer 38

1) Same protein in a different crystal form
2) Same protein bound to a ligand or another protein
3) Same protein in a slightly different conformation
4) Related protein (homologue from a related species)
5) A complex composed of more than one protein, with known structures for each component.

Question 39

What is a requirement of a protein in order to use SAD to find its phase?

Answer 39

It must have a methionine for every 100-150 residues.

Question 40

How do SAD and MAD work?

Answer 40

1) proteins are grown in a minimal media containing selenomethionine
2) X-rays are shined on both selenomethionine protein and native one
3) Compare the diffraction patterns of the two x-ray diffraction images.
4) Use the differences between the two to determine the location of the heavy atoms and determine structure form there.

Question 41

How do SIR and MIR work?

Answer 41

1) protein crystals soaked with a small number of heavy atoms (gold, mercury, platinum, lead) that bind to the surface cysteine and histidine residues.
2) differences in scattering contribute to

Question 42

What are some parameters that can be adjusted during the refinement stage of a model?

Answer 42

1) Atomic coordinates (x,y,z) for each atom
2) Temperature factors/ B factors which describe the atomic motion usually between 5-80 Angstroms
3) Occupancy (how often does a flexible chain/ ligand occupy a region of the unit cell?)

Question 43

What is R-factor?

Answer 43

A measurement of the agreement between calculated and observed diffraction patterns. The smaller it is the more accurate the model.

Question 44

Why would even a good crystal, data and model not have an R-factor of 0?

Answer 44

• Protein channels have large channels of water are not included in the structure but are affecting the actual protein.
• Disorder and vibration are seen in proteins which are not accounted for in the model

Question 45

What is an issue with using R-factors to assess how good a mode is?

Answer 45

The refinement process creates bias since the atomic model is used along with the diffraction pattern to calculate the electron density.

Question 46

How does the R-free model work?

Answer 46

Before the refinement process, 5-10% of the experimental observations are removed from the data set. The, refinement is performed using the remaining 95-90% (R-work). The R-free value is calculated based on how well the model predicts the 5-10% that was not used for refinement.

R-free value is usually a little higher than the R-work value, but the difference between them should be less than 5%.