JW - Protein Architecture III

Question 1

What is Cryo-EM? (2)

Answer 1

Flash freeze samples ( eg liquid ethane)

• For water to go into glass like structure (not ice)
• Preserve structure of object in aqueous environment but limit radiation damage

Question 2

Why is rapid freezing crucial in the process?

Answer 2

Need rapid freezing process to prevent water from entering into crystalline form as this results in scattering

Question 3

What are 3 ways to enhance signal-to-noise and resolution in cryo EM?

Answer 3

1) EM crystallography (3D and 2D crystals possible)

• Regular lattice of objects allows averaging over many particles as well as using the periodicity of the objects to enhance resolution (diffraction of electrons leads to diffraction pattern)

2) Single particle reconstruction

• Accumulate many images of individual particles to improve signal to noise

3) EM tomography:

• Rotate object and record a series of (2D images, projections)
• Reconstruct 3D object from

Question 4

How does scattering result in interpretation of molecular shape? (4)

Answer 4

1. Incident radiation on sample solution
2. Using a detector, measure angle of scattered radiation
3. Present on a graph: l(q) intensity of radiation against angle (q)
4. l(q) depends on the shape of the scattering molecule

If one knows the individual domains with atomic detail, one can arragne them to give the right shape

Question 5

What information does Small Angle X-ray Scattering (SAXS) provide?

Answer 5

Small Angle X-ray Scattering (SAXS) is an analytical technique giving access to information about the structure of materials at the nano- and meso-scale

1. P(r) distribution (gives size and shape idea)
   Pair correlation function, P(r), is Fourier Transform of I(q)
2. Kratky plot (if not going to 0 it must be bound)

Question 6

What is the process of structure determination by X-ray crystallography? (4)

Answer 6

1) Make (protein) crystals

2) Collect diffraction data

3) Convert diffraction data into electron density map

• Needs information in addition to diffraction spots
  Phasing problem;

4) Fit molecular model into electron density map

Question 7

The art of making protein crystals (2)

Answer 7

1. Make supersaturated protein solution
2. Find buffer conditions that encourage crystal growth over precipitation
   i.e. screen many buffer conditions

Question 8

What are the 3 different techniques to convert the diffraction pattern into an electron density map?

Answer 8

• Make additional crystals with heavy atom derivatives
• Incorporate seleno-methionine protein using its anomalous diffraction to determine the phases
• If a similar protein structure is known to back-calculate the electron density map

Question 9

What is Nuclear Magnetic Resonance (NMR), and how does it work?

Answer 9

NMR involves the transition between high and low-energy spin states of atomic nuclei

• particularly protons (1H), carbon (13C), and nitrogen (15N), providing information about molecular structure