X-ray crystallography and crystals

Question 1

What is X-ray crystallography (XRC)?

Answer 1

1. One of the best method for visualising
2. A way of taking ‘photographs’ of molecules, but using X-rays.
3. Electrons from atoms scatter the X-rays, so we see the electron cloud around the molecule –we build a model of atom positions to interpret the image.

Question 2

Describe fundamental principle of diffraction for XRC

Answer 2

1. In order to see a detail (x) meters in extent, the illuminating radiation you use must have a wavelength at most double that size
2. So protein molecules are invisible to visible light.
   4, Even a concentrated solution of protein is transparent
3. Light passes without interacting, so no information on the protein is encoded in the emission

Question 3

What is the wavelength range of an x-ray

Answer 3

1. Wavelength range from 0.1-100 Å

2. 10-8 cm = 1 Å

Question 4

What is used as the ruler in XRC

Answer 4

1. Crystallographers measure distances between atoms in Å
2. To measure atomic distances through interference and to determine the structure of molecules, our “ruler” must have atomic dimensions
3. Perfect “rulers” to measure Å distances are X-rays
4. X-rays used by crystallographers are ~ 0.5 to 1.5 Å
5. Just the right size to measure the distance between atoms in a molecule
6. They are close to inter-atomic distance

Question 5

How to build an atomic microscope

Answer 5

1. For small objects (molecules) need short wavelength waves
2. Inter-atomic distances are 1.2 – 2.0 Å so suitable wavelengths are < 2 Å (0.2 nm)
   - X-rays
   - electrons
   - neutrons

Question 6

How does a microscope work

Answer 6

1. Light strikes the object and is diffracted in various directions
2. The lens collects the diffracted rays and reassembles them to form an image

Question 7

Can you form an x-ray microscope

Answer 7

1. Can’t build an X-ray microscope
2. No X-ray lens
3. Lens focuses visible light, but the refractive index for very short wavelengths is ~ 1, so far no material can be used to focus X-rays
4. With X-rays, we can detect diffraction from molecules, but we need a different approach to reassemble the image

Question 8

Describe how X-ray crystallography works

Answer 8

1. High-powered X-rays are aimed at a tiny crystal containing trillions of identical molecules
2. Crystal scatters the X-rays onto an electronic detector
3. Like a disco ball spraying light across a dance floor
4. Electronic detector similar to those used to capture images in a digital camera

Question 9

What are the steps in solving a protein structure by X-rays

Answer 9

1. Purify protein (10 mg) or more
2. Grow a crystal
3. Collect and process diffraction
4. Phase the diffraction data
5. Calculate an electron density map
6. build and refine the structure

Question 10

What do you need to know about the protein before the purification stage

Answer 10

1. Sequence, molecular weight
2. Disulphide bonds, glycoprotein?, phosphorylated?
3. Maximum stability/activity, degradation?
4. Cofactors
5. Tags used in purification
6. Secondary structure prediction- If only secondary structure will be difficult to analyse
7. Homologs with known structure

Question 11

What makes a good protein sample

Answer 11

1. Pure- SDS-PAGE, Mono Q, Iso Electric Focusing, mass spectroscopy
2. Defined buffer
3. Defined concentration
4. No aggregation
   - Dynamic light scattering, size exclusion
   - To improve: salt, pH, temperature, detergent, batch, cofactors, binding partners, mutagenesis
5. Need lots of pure protein- (bacterial/yeast/insect/mammalian expression)

Question 12

What is SDS-PAGE

Answer 12

1. SDS-PAGE is an electrophoresis method that allows protein separation by mass.

Question 13

What is Mono Q

Answer 13

1. Mono Q are strong anion exchange chromatography columns for protein analysis or small scale, high resolution polishing of proteins.

Question 14

What does a protein used in XRC look like

Answer 14

1. typically about 1-5 mg for about 2000 conditions
2. screen highest purity achievable.
   - Affinity, ion exchange and gel filtration, chromatography is a common protocol.
3. affinity tags should be minimal or cleavable with specific proteases
4. typically requires several (many) constructs

Question 15

Why are crystals used

Answer 15

1. X-ray scattering from a single molecule would be unimaginably weak and could never be detected above the noise level (scattering from air and water)
2. A crystal arranges huge numbers of molecules in the same orientation, so that scattered waves can add up in phase and raise the signal to a measurable level
3. A crystal acts as an amplifier!

Question 16

What is a crystal

Answer 16

1. Under certain conditions, molecules line up into a regular grid (a “lattice”).
   - Example: table salt
2. Under certain conditions, entire proteins will pack into a regular grid (a lattice)
   - Proteins can form crystals

Question 17

What are the components of a crystal

Answer 17

1. Molecule sits in small box- unit cell

2. Unit cells line up in 3D

Question 18

What is the symmetry in packing of molecules (in crystals)

Answer 18

1. Mirror symmetry
2. Rotation symmetry
3. Other types of symmetry operation are reflection and inversion.
4. Because macromolecules (protein and nucleic acid) are chiral, macromolecular crystals cannot contain these symmetry elements.

Question 19

Describe crystal lattices

Answer 19

1. Periodic arrangement in 3-dimensions
2. A crystal unit cell is defined by its cell constants and is the building block for the whole crystal
   - Edges: a, b, c
   - Angles: a, b, g
3. 14 lattice crystal arrangements

Question 20

What are space groups and how many are there

Answer 20

1. A point group describes the symmetry of a finite object
2. A lattice defines the translational symmetry
3. Combining point group and Bravais lattice symmetries generates space group symmetry (but additional symmetry elements involving a translation need to be considered).
4. The space group is a complete description of the symmetry of an (ideal) crystal. (Ideal implies infinite !)
5. Knowing the space group, and the contents of the asymmetric unit, defines the positions of all atoms in the crystal.
6. There are a total of 230 different space groups (first derived in nineteenth century), of which only 65 are possible for chiral molecules.

Question 21

Why do we need to know how proteins form crystal lattices?

Answer 21

1. Because when X-rays interact with crystals, coherent diffraction occurs from planes in the crystal
2. The resulting diffracted waves will be the sum of diffraction from interaction with matter all the way along the plane
3. This set of ‘planes’ will give rise to 1 diffraction spot
4. As the spots are diffraction from all of these parts of the crystal, their signal is stronger than if we only had 1 row of ducks or one duck
5. Darker spots on the detector (from amplified diffraction waves) can be measured more reliably.

Question 22

What are problems with using a crystal

Answer 22

1. Sometimes a protein will adopt a different structure in a crystal than it does in its natural environment
2. Crystallography gives you a static snapshot of a protein’s structure
3. Usually (but not always) this snapshot corresponds to the protein’s “time and space average” structure in 3D