Determination of Protein Structure

Question 1

What is the primary technique for determining 3D structures, ranging from salt to whole virus particles?

Answer 1

X-ray crystallography

Question 2

What form does the sample need to be in for X-ray crystallography?

Answer 2

Crystals

Question 3

What proportion of structures in the PDB archive has X-ray diffraction techniques solved?

Answer 3

Over 90%

Question 4

Give examples of the achievements of crystallography in the last 50 years

Answer 4

The structure of DNA has shown how genetic information is stored, replicated and used
Cro repressor-operator complex shows how proteins can interact with DNA and control the regulation of gene expression

Question 5

What have elucidating the structures of enzymes shown?

Answer 5

How biochemical processes are catalysed

Question 6

What have elucidating the structures of virus particles shown?

Answer 6

How they assemble

Question 7

What have elucidating the structures of proton-translocating ATPase shown?

Answer 7

How the proton gradient is used to make ATP

Question 8

What can protein crystallography be used for?

Answer 8

Structure-based drug design, development and improvement

Question 9

What happens when waves impinge on a body?

Answer 9

They are scattered and converted into an image by means of a lens

Question 10

Using waves for large objects works fine but what are they limited to?

Answer 10

About the wavelength of radiation used

Question 11

What do we want to resolve about molecules?

Answer 11

The distances between atoms

In Angstroms

Question 12

What is 1 Angstrom equal to?

Answer 12

E-10 metres

Question 13

When is the resolution limit reached?

Answer 13

When two point-like objects can not be imaged as two distinct images

Question 14

What type of waves are needed to resolve atoms?

Answer 14

X-rays

Question 15

Is visible light far too long or short to resolve atoms?

Answer 15

Far too long

Question 16

X-rays interact with matter weakly, true or false?

Answer 16

True

Question 17

What are X-rays scattered by?

Answer 17

Electrons and so heavier atoms with more electrons scatter more

Question 18

When were X-rays discovered and who by?

Answer 18

8th November 1895

Wilhelm Rontgen

Question 19

Do X-ray lenses exist?

Answer 19

No

Question 20

What is a crystal?

Answer 20

A repeating ordered array (LATTICE) of molecules in 3D in which the molecules are identically oriented

Question 21

How many molecules will a typical protein crystal of 0.3mm in each direction have in each dimension?

Answer 21

E5 molecules in each dimension which is about E15 molecules

Question 22

What is the total scattering a sum of?

Answer 22

The individual scatterers, or one thousand million million times stronger than the individual molecule

Question 23

Scattering from a crystal is concentrated in discrete directions determined by what?

Answer 23

The crystal lattice

Question 24

What does the fourier transform relate?

Answer 24

Te sum of all the reflections to the electron density at all points in the crystal repeating unit

Question 25

Essentially, what does the fourier transform tell us?

Answer 25

What mixture of sine-waves is required to make up any function

Question 26

What information about a wave is needed for the fourier transform?

Answer 26

Phase information

Question 27

What techniques can you use to get phase information of a wave?

Answer 27

Molecular replacement Multiple isomorphous replacement Anomalous dispersion

Question 28

What can we calculate from diffraction data and phases?

Answer 28

Electron density to get an atomic model

Question 29

What can we calculate from an atomic model?

Answer 29

Electron density and from this diffraction data and phases

Question 30

What is the iterative process called?

Answer 30

Refinement and rebuilding

Question 31

What can the difference/omit map be used for?

Answer 31

Finding ligands

Question 32

What affects resolution?

Answer 32

The maximum scattering angle observed for the data

Question 33

What is the primary way that stereochemistry of a model can be assessed?

Answer 33

Using a Ramachandran plot

Question 34

Protein crystals need to be obtained gently, true or false?

Answer 34

True

Question 35

How are protein crystals generated?

Answer 35

By removing the protein very slowly out of solution

Question 36

What factors affect protein solubility?

Answer 36

Concentrations of salt/precipitate pH Temperature

Question 37

Name a method for obtaining crytals

Answer 37

Vapour diffusion

Question 38

Crystals are easy to grow, true or fasle?

Answer 38

False, they need many different conditions testing and optimising

Question 39

What are the mechanics of X-ray data collection?

Answer 39

The crystal is oscillated about a small angle and the resultant diffraction recorded