BB8 Structures From Crystallography

Question 0

Crystallography works because

Answer 0

• crystals have building blocks of packed and orientated molecules
• the x-rays interact with the electrons from these molecules

Question 1

Crystallography

Answer 1

• can reveal the atomic structure of macromolecules

* requires crystals and x-rays

Question 2

Crystallization

Answer 2

• slowly changing the solution environment around a protein to make it less soluble
• eg “slow salting out”

Question 3

Slow salting out

Answer 3

• salts are added to a pure protein solution

* the right concentration induces the protein to crystallize but not precipitate

Question 4

Crystals

Answer 4

• should be 1mm maximum in any dimension

* water must always be present in protein crystals – must not dry out (30-80% water)

Question 5

Laboratory X-ray source

Answer 5

• usually a Copper X-ray tube
• Cu used as a target for electrons
• produces x-rays with characteristic wavelength λ = 1.54Å

Question 6

Another X-ray source

Answer 6

• synchrotron radiation
• electrons accelerated in a ring of magnetic
• emit electromagnetic radiation – X-rays

Question 7

When X-rays strike protein crystal

Answer 7

• some pass straight through
• some interact with the molecules in the crystal
(scatter in numerous specific directions – diffraction)

Question 8

Diffraction pattern

Answer 8

• total scattering pattern

* recorded as a # of spots by an electronic detector

Question 9

Each spot from diffraction

Answer 9

• is a scattered wave of X-rays
• has unique intensity
• contains info about ALL the atomic positions in the protein molecules

Question 10

Fourier transform

Answer 10

• math technique used to obtain the atomic positions of the molecules in X-ray crystallography
• needs the phase information for each unique spot

Question 11

Phase information of each spot

Answer 11

• position of the crest and trough of the scattered wave of each diffraction spot relative to other spots
• lost in X-ray crystallography = the phase problem
• extra techniques to gain back this phase information

Question 12

After diffraction pattern and fourier transform, next step in X-ray crystallography is to

Answer 12

calculate an electron density map
• high density = where atoms ARE
• low density = where atoms ARE NOT
• ultimately a protein structure can be produced

Question 13

The amount of detail obtained in X-ray crystallography depends on the

Answer 13

resolution – how easy it is to distinguish features within the structure

Question 14

At 6Å resolution

Answer 14

• overall course of the chains only

Question 15

Between 4.0 and 2.8 Å resolution

Answer 15

• groups within the protein structures can be determined

* density where side chains might be

Question 16

Between 2.5 and 2.0 Å resolution

Answer 16

• reliable positions for the side chains of residues

* typically protein structures are solved around 2.0 Å

Question 17

Between 1.5 and 1.0Å resolution

Answer 17

• individual atoms can clearly be seen

Question 18

When proteins are crystalline, they are

Answer 18

• usually in their native conformations

* proven when enzymes were shown to be still active as crystals

Question 19

Potential problems

Answer 19

• structures are STATIC, not dynamic

* potential for distortion through crystal contacts

Question 20

Circular Dichroism (CD) spectroscopy

Answer 20

• CD spectra measure how proteins interact with circularly polarized light
• absorption effect

Question 21

… structures in proteins have specific CD features

Answer 21

secondary

Question 22

… of a protein consists of different amounts of these secondary structure features

Answer 22

• native conformation

Question 23

CD can be used to see if a protein

Answer 23

• is in its native conformation indifferent solution conditions

Question 24

CD spectrum from a protein is a combination from these features

Answer 24

``` CD(protein) = • %CD (α-helix) + • % CD (β-sheet) + • %CD (β-turn) + • %CD (“other”) ```

Question 25

CD is a very sensitive technique for noting changes in the

Answer 25

• percentages of secondary structures

Question 26

If a protein CD spectrum in a given solution differs from the spectrum in physiological conditions the protein

Answer 26

• is no longer in its native conformation

Question 27

Extended form of CD

Answer 27

Synchrotron Radiation Circular Dichroism (SRCD) • more powerful light source • extended lower wavelength range (light blue area) • more information from the spectrum data

Question 28

Diffracted x-rays pattern gives info about structure

Answer 28

1. Electrons scatter x-rays - amplitude of wave scattered proportional to number of electrons 2. The scattered waves recombine - reinforce one another if in phase, cancel one another if out of phase 3. The way in which the scattered waves recombine depends only on the atomic arrangement

Question 29

X-ray with array of spots = reflections

Answer 29

Wave with amplitude proportional to the sqrt of intensity Used to reconstruct image with Fourier transform to make an electron density map

Question 30

Electron density map

Answer 30

3D representation of where the electrons are most densely localized • used to determine positions of atoms in crystallized molecule

Question 31

Resolution

Answer 31

* used to interpret electron density map * determined by the number of scattered intensities used in Fourier transform * ultimately determined by degree of perfection of crystal