DLS and SAXS

Question 1

What is Rayleigh Scattering?

Answer 1

When light hits small particles it scatters in all directions.
Particles must be small compared to the wavelength
Some wavelengths scatter more than other

Question 2

What is observed in the scattering intensity of a laser?

Answer 2

Time dependent fluctuation
Lasers are monochromatic and coherent

Question 3

Why do you observe a time-dependent fluctuation with a laser?

Answer 3

Because the small molecules are undergoing Brownian motion.
Therefore the distance between scatters is constantly changing

Question 4

Scattered light then undergoes either constructive or destructive _ due to the surrounding particles

Answer 4

Interference
There is intensity fluctuation within this

Question 5

What is Mie Scattering?

Answer 5

• Scattering of light or other electromagnetic radiation by particles with similar size or larger than the wavelength of the light
• White light scattered in all directions.

Question 6

What occurs in opalescent glass and blue eyes?

Answer 6

Rayleigh scattering
Greater for blue light than any other

Question 7

What is Classical or Static Light Scattering?

Answer 7

Constant measuring of scattering (static)
Measured at various angles

Question 8

Purposes of Static Light Scattering

Answer 8

• Direct measure of molecular mass
• Determine oligomeric state of protein
• Determine that mass of aggregates

Question 9

What is dynamic light scattering

Answer 9

Measuring scattering at one angle
Scattering is observed to be changing over time
Also known as photon correlation spectroscopy or quasi-elastic light scattering

Question 10

Purposes of DLS

Answer 10

• measures rate of diffusion
• Determines “stroke radius” or hydrodynamic radius
• Senses very small amounts of aggregated protein

Question 11

Do emulsions and molecules in suspension undergo Brownian motion?

Answer 11

Yes
This is the motion induced by the bombardment by solvent molecules that themselves are moving due to their thermal energy

Question 12

What does the decay time of light scattering fluctuations tell us?

Answer 12

The diffusion coefficient of the particles
Inversely proportional

Question 13

How to we obtain the decay time?

Answer 13

From the time-dependent correlation function of the scattered light.

Question 14

How do we determine particle size?

Answer 14

D=(kB T)/6πRη

• D: diffusion constant
• R: hydrodynamic radius
• k: Boltzmann-constant
• T: temperature
• η0: solvent viscosity

Question 15

Larger particles move _

Answer 15

Slower

Question 16

Smaller particles move _

Answer 16

Faster

Question 17

How does DLS work?

Answer 17

• A beam of laser light is focused in the sample
• Particles in the scattering volume scatter light in all directions
• The scattered photons are measured by a photomultiplier tube
• The Intensity appears to fluctuate randomly
• A digital correlator is used to compute the autocorrelation function

Question 18

What is the autocorrelation function?

Answer 18

It appears as a signal with exponential decay
The bigger the particle the longer the correlation lag time.
So small particles will look like skate ramps (concave) and larger ones will look more convex
Lots of fluctuation = small particle.

Question 19

What is Pd?

Answer 19

Polydispersity
How much of the proteins formed polymers
More than 20% Pd means that the peak will broaden

Question 20

What does DLS actually measure?

Answer 20

The hydrodynamic radius

The translational diffusion coefficient will depend not only on the size of the particle “core”, but also on any surface structure

Question 21

Requirements of DLS (4)

Answer 21

• The particles being measured are ‘spherical’
• The suspension is dilute, such that the scattered light is measured before it is re-scattered by other particles (backscattering).
• The optical properties of the particles and the medium surrounding them is known
• The particles are homogeneous

Question 22

Applications of DLS

Answer 22

• Protein Crystallography - To check before the crystallization occurs
• Small angle X-ray diffraction
• Nanotechnology
• Medicine

Question 23

What is Small-Angle X-ray Scattering?

Answer 23

A low-resolution technique that provides high-precision information on size and shape
Useful for flexible systems
Not the same as an X-ray

Question 24

SAXS is ideal for the characterisation of _

Answer 24

o multidomain proteins (don’t like to crystalize)
o multisubunit complexes
o flexible systems

Question 25

Scattering can be done with, _, _ or _ radiation

Answer 25

X-ray, UV or Infrared radiation.

Question 26

In X-ray diffraction, crystals have a regular structure with a repeat distance that is about the same as the _

Answer 26

Wavelength

Question 27

What causes the diffraction pattern in X-ray diffraction?

Answer 27

X-ray interference between wavelengths scattered by atoms within regularly spaced protein in the crystal

Question 28

What is the theta angle?

Answer 28

The angle of diffraction - measured in degrees For practical reasons the diffractometer measures an angle twice that of the theta angle – “2-theta”

Question 29

What can we gain from diffraction patterns?

Answer 29

Can use to calculate electron density map Position of the reflections tells us something about symmetry Often see a dark "water ring"

Question 30

What can we see at 5Å and 1Å resolution?

Answer 30

At low resolution, 5Å or higher one can obtain the shape of the molecule and sometimes identify alpha-helical regions as rods of electron density. At 1Å resolution one sees atoms as discrete ball of density

Question 31

Why is small angle scattering observed in molecules in crystals and in solution?

Answer 31

It arises from the coherent secondary wavelets that are scattered by atoms within a single molecule

Question 32

Stages of preliminary characterisation

Answer 32

* Purity - SDS page * Concentration - Absorbance * Monodispersity - DLS * Matched solvent - dialyse sample

Question 33

How to calculated scattering factor?

Answer 33

|s|=4π sinθ/λ = scattering factor Measure angles for solution then subtract angles from solvent to get scattering factor.

Question 34

Intensity of scattering becomes _ further from the beam stop.

Answer 34

Less

Question 35

Stages of data validation

Answer 35

* Initial inspection - Guinier plot * radiation damage - measure time course * concentration effects * standards - measure water and/or secondary protein standard

Question 36

What is the main sign of radiation damage?

Answer 36

If you measure the signal multiple times and it changes Reducing conditions can prevent this

Question 37

What is an indication of an aggregated protein in SAXS?

Answer 37

Steep line in sample-solvent graph, for example graph starts with concave curve.

Question 38

What does an SAXS log plot tell us?

Answer 38

Shape and size

Question 39

What does a Guinier plot tell us?

Answer 39

Rg, MM plots Ln I(s) against s^2 Knowing MM can determine if 24-mer or 48-mer etc

Question 40

What does a P(r) plot tell us?

Answer 40

Diameter Usually 1 peak Maximum y = maximal diameter on x Can be used to find conformational change

Question 41

What does a Porod plot tell us?

Answer 41

Volume (s)*s^4

Question 42

How can you use SAXS to determine a lo-res 3D model?

Answer 42

Ab initio shape determination Fill sphere with compact dummy atoms and change their orientation until they match the data for your sample