High Resolution Techniques

Question 1

High resolution techniques

Answer 1

Nuclear Magnetic Resonance (NMR)
Cryogenic Electron Microscopy (Cryo-EM)
X ray Crystallography of Biomacromolecules
Mass spectrometry

Question 2

Nuclear Magnetic Resonance Spectrocopy

Answer 2

Objective: For structure determination

Principle:
Proton resonance frequency = intrinsic property
Exposed to radio frequencies, absorb them and enter higher energy state. Emit radio frequency and return ground state. NMR detects frequency & instensity of emitted radio frequency.

Proton resonace frequency infleunced by local environment (shielding effect: e shields protons). Affect chemical shift

Component
1H, 15N, 13C, 19 F, 31P (Unpaired proton)

Question 3

1H 15 N HSQC

Answer 3

Objective: structure of protein
Principle
Heteronuclear Single Quantum Correlation: Look at N and H
Gives peaks. Each peak represents unique NH bonds in its environment.
Components
Need 13C, 1H, 15N

Question 4

Cryogenic Electron Microscopy (Cryo- EM)

Answer 4

Objective: Protein Structure

Principle: Froze sample

Steps:
Solution of target protein is placed on grid & frozen in liquid nitrogen.
Each grid contains snapshot of target protein
Grid exposed to beam of e.

Results: pattern of electron densities. Model building

Question 5

X-ray Crystallography of Biomacromolecules

Answer 5

Objective: Dtermine 3D structure of protrins & protein-ligand complexes

Principle: Crystallyze target protein

Steps: Crystal, Xray beam to obtain diffraction pattern, computational programs to obtain electron density map, computational programs to obtain protein model.

Results: Electron density map to compute protein structure.

• Diffraction pattern: address of all atoms
• Electron density map: location of aa

Question 6

Mass spectrometry

Answer 6

Objective: determine strcutural changes of target protein

PTMs
Complexes