This document provides an overview of structural biology, including its focus on determining the molecular structures of biological macromolecules like proteins and nucleic acids. It discusses key developments in the field, such as Watson and Crick's discovery of DNA's structure, and the two main experimental approaches used today: X-ray crystallography and nuclear magnetic resonance spectroscopy. X-ray crystallography involves crystallizing a protein and analyzing its diffraction pattern to determine structure, while NMR studies protein structures in solution using magnetic fields. Both techniques have benefits and limitations related to the size of molecule that can be studied and experimental requirements.

1. TOPIC 1
OVERVIEW OF STRUCTURAL BIOLOGY

2. What is it?
• a branch of molecular biology, biochemistry,
and biophysics concerned with the molecular
structure of biological macromolecules,
especially proteins and nucleic acids, how they
acquire the structures they have, and how
alterations in their structures affect their
function.

3. Why is it important?
• Function of biological macromolecules is
intricately related to their three-dimensional
shape and structure. Structural knowledge is
therefore an important step in understanding
function.

4. Protein structure

5. Some Landmarks in Macromolecular Structure Determination
Watson and
Crick
Perutz and
Kendrew
Hodgkin
Pauling
Great ideas have always faced violent opposition from mediocre minds
-Albert Einstein
Hodgkin

6. Some Landmarks in Macromolecular Structure
Determination……..contd.
Photosynthetic reaction centre
Potassium channelVirus
Virus Potassium channel

7. DNA : Diffraction pattern

8. Tertiary protein structure: protein folding
Three main approaches:
[1] experimental determination
(X-ray crystallography, NMR)
[2] Comparative modeling (based on homology)
[3] Ab initio (de novo) prediction
(Dr. Ingo Ruczinski at JHSPH)

9. Experimental approaches to protein structure
[1] X-ray crystallography
• Used to determine 80% of structures
• Requires high protein concentration
• Requires crystals
• Able to trace amino acid side chains
• Earliest structure solved was myoglobin
• Solubilization of the over-expressed protein
• Obtaining crystals that diffract
• Structure determination by diffraction of protein crystals
• Size of a molecule: no theoretical limit

10. [2] NMR
• Magnetic field applied to proteins in solution
• Largest structures: 350 amino acids (40 kD)
• Does not require crystallization
• Solubilization of the over-expressed protein
• Structure determination of a molecule as it exists in
solution
• Size-limit is a major factor

11. Principles of X-ray crystallography
• Crystals act as a three-dimensional grating and produce diffraction
• The diffraction pattern contains complete information on the placement of
scatterers (electrons in atoms)
• By fourier transforming the diffraction pattern, we can obtain information
on the structure of the molecule in the crystals

12. Principles of NMR
• Measures nuclear magnetism or changes in nuclear magnetism in a molecule
• NMR spectroscopy measures the absorption of light (radio waves) due to
changes in nuclear spin orientation
• NMR only occurs when a sample is in a strong magnetic field
• Different nuclei absorb at different energies (frequencies)

13. X-ray versus NMR
• Producing enough protein for trials
• Crystallization time and effort
• Crystal quality, stability and size
control
• Finding isomorphous derivatives
• Chain tracing & checking
• Producing enough labeled protein
for collection
• Size of protein
• Assignment process is slow and
error prone
• Measuring NOE’s is slow and
error prone
X-ray NMR

14. Steps in obtaining a protein structure
Target selection
Obtain, characterize protein
Determine, refine, model the structure
Deposit in database

15. X-ray crystallography
http://en.wikipedia.org/wiki/X-ray_diffraction
Sperm Whale Myoglobin

18. Nuclear magnetic resonance spectroscopy