TOPIC 1

OVERVIEW OF STRUCTURAL BIOLOGY

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.

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.

Some Landmarks in Macromolecular Structure Determination

Watson andCrick

Perutz andKendrew

Hodgkin

Pauling

Great ideas have always faced violent opposition from mediocre minds-Albert Einstein

Hodgkin

Some Landmarks in Macromolecular Structure Determination……..contd.

Photosynthetic reaction centre

Potassium channelVirusVirus Potassium channel

DNA : Diffraction pattern

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)

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

[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

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

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)

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

Steps in obtaining a protein structure

Target selection

Obtain, characterize protein

Determine, refine, model the structure

Deposit in database

X-ray crystallography

http://en.wikipedia.org/wiki/X-ray_diffraction

Sperm Whale Myoglobin

Nuclear magnetic resonance spectroscopy