x ray pt 1

Question 1

what did ramakrishnan, rhomas a steitx and ada yonath win the nobel prize for and when

Answer 1

2009
for studies of the structural function of the ribosome

Question 2

what did robert lefkowitz and brain kobika win the nobel prize for and when

Answer 2

2012 and for studies of G protein coupled receptors

Question 3

x ray crystallography

Answer 3

method that produced the highest number of nobel prizes in chemistry and in physiology or medicine

one of the three methods that allow us to see biological macromolecules- want to see them bc form follows function

Question 4

structure detemrines function

Answer 4

the structure and function of all things are intimately related and cannot be separated

if you know the structure of a protein, you can understand its function
knowing their structure was critical for our discussion of different proteins

Question 5

structural biology

Answer 5

the study of molecular structure and dymanics of biological macromolecuoes and how alterations in thier structures affects thier function

Question 6

what are the four ingredients of seeing

Answer 6

object
light (visible light, lambda= 300-740 nm)
lens to refocus scattered light (camera, human eye)
recording/ displaying device (film, retina)

Question 7

why cant we see proteins

Answer 7

because theyre smaller than the wavelength of visible light so we need x rays

Question 8

to visuallize an object

Answer 8

light has to scatter off that object
But light does not interact with objects smaller than its wavelength
⇨ Light is a wave!

Question 9

wavelength of x rays

Answer 9

0.1-0.2 nm

Question 10

what is another probelm for using x rays

Answer 10

no lens can refocus x rays

Question 11

what is the solution to the x ray lens problem

Answer 11

use a computer as a lens

X-rays diffracted from the object are measured by a detector
- Measurements are fed to a computer, which reconstructs and
display the image of the molecule

Question 12

what is the third problem for x rays

Answer 12

X-ray scattering from a single protein molecule is
weak and difficult to detect

Question 13

solution to the third problem

Answer 13

• A crystal arranges a large number of molecules in the same orientation
  → provides a sufficient amount of material to scatter the incoming X-ray
• Scattered waves add up in phase and raise the signal even more

Question 14

the signal is raised in __

why?

Answer 14

discrete patterns

because scattered waves that are not in phase cancel each other as shown by thomas young’s double slit experiment

protein crystal diffraction patterns are a bit more complicated

3D not 2D, millions to billions of repeating units (not just two)

Question 15

what is a crystal

Answer 15

A solid material whose constituents (atoms, ions, or molecules)
are arranged in an ordered, periodic pattern that extends in all
directions
composed of unit cells- the basic repeating unit= a crystal can be generated from the unit cell by copy and translation

Question 16

what constitutes a unit cell?

Answer 16

“Asymmetric units”, the smallest repeating unit that forms the
crystal ⇒ the part of the unit cell which by copy, translation, and
rotation can generate the complete unit cell

an asymmetric unit contains one or more molecules of the same proteins

Question 17

a PDB file describes

Answer 17

the contnet of a given crystals asymmetric unit

Question 18

what is inside an asymmetric unit

Answer 18

protein molecule(S), water, ions, small molecules

electron cloud!! makes the x rays scatter
x rays dont scatter off the nucleus

Question 19

everything inside the a.u and therefore the entire crystal can be described as

Answer 19

propability function- describes the propability of an electron being present at a specific 3D coordinate

p(x,y,z)

Question 20

x rays scatter off the electrons in the crystal are recorded as

Answer 20

2D diffraction images (a collection of dots or reflections)

Question 21

what are the two consequences of X ray being a 3D phenomenon

Answer 21

Two consequences:
1. A reflection can be described by its coordinate in this 3D space ℎ, +, ,
2. Have to move around the detector to collect as many data points
(reflections) as possible ⇨ for practical reasons we move the crystal

Question 22

what are the two 3D spaces

Answer 22

real space
reciprocal space

Question 23

real space

Answer 23

crystal
electron cloud
expressed by the function p(x,y,z)

Question 24

reciprocal space

Answer 24

summed up (interfered) waves of diffraction x rays (i.e. reflections)

expressed by F(h,k,l) values

called “structure factors” which describe the intensity of a given set of reflections

Question 25

what is the relationship between real space and reciprocal space

Answer 25

can be described by fourier transform

Question 26

protein crystals are

Answer 26

precipitated protein molecules

random precipitation results in amorphous structures

crystals are ordered i.e. protein molecules are arranged in the same orientation

Question 27

how do we crystallize proteins

Answer 27

Similar to how salt or sugar
crystals are formed
- From supersaturated solutions
But we cannot boil protein
samples, so instead, we add
precipitants
→ Take away water molecules in
the protein hydration shell
⇨ Forces protein molecules to
come together and associate with
each other
(Hopefully in an ordered manner!

Question 28

what techniques can we use to further promote crystal growth

Answer 28

vapour diffusion
liquid liquid diffusion
dialysis
experiments in space

principle is essentially the same for all methods- allows protein hydrophobic patches come together and bind

Question 29

what method to further promote crystal growth is used

Answer 29

vapour diffusion

Question 30

what is happening in the drop in vapour diffusion

Answer 30

can be depicted in a phase diagram

1. When solution gets
   concentrated enough
   nucleation occurs
2. Initial crystal growth pulls
   some protein out of solution
3. With decreasing [protein]
   solution is back into
   metastable range (where
   crystal growth is optimal)
   ⇨ Grows only a few large crystals

Question 31

how do we know what kind of and how much percipitant to use

Answer 31

Crystallization condition is protein-dependent
E.g.,
- highly conserved homologous proteins
- same protein with a few mutated amino acid residues
- same protein with a different ligand
will all likely crystallize in a different crystallization condition
⇨ We have to find favourable conditions by trial-and-error

Question 32

crystal screens

Answer 32

Assortment of conditions of salts,
buffers, pH’s, and additives
commercially available
- E.g., best conditions from the literature
* Very often initial hits from the
screen(s) produce small, poor-
quality crystals
* Optimization of conditions
typically required

Question 33

factors affecting crystal growth (most important)

Answer 33

ionic strength
protein concentration
pH
temperature
monodispersion
purity of protein