The Structures of Atoms Found in Organisms

Different Types of Covalent Bonds

the shared electrons glue the atoms together

Basic Structure of An Amino Acid

alanine arginine asparagine aspartate cysteine

glutamate glutamine glycine histidine isoleucine

leucine lysinemethionine

phenylalanine histidine

serine threonine tryptophan tyrosine valine

The Twenty Amino Acids

Proteins are Polymers of Amino Acids

some proteins in your body have the same amino acid sequence as those found in the chimpanzee

Critical Stages in an X-ray Analysis

• Cloning, over-expression, and protein purification

• Crystallization

• Data collection and processing

• Interpretation of electron density maps

Fundamentals of Protein Crystallization

1. Crystallization is simply achieved by reducing the solubility of the protein under conditions where the stable state becomes the crystalline lattice.

2. The problem is finding those conditions!

3. Must determine the appropriate precipitant and state of the protein for crystallization to occur.

Finding Crystallization Conditions

1. This is still a matter of trial and error.

2. There is no guarantee that a given protein can be crystallized.

3. Crystallization screens have been developed that improve the chances of finding suitable conditions.

4. A large number of crystallization screens are commercially available from Hampton Research.

Important Parameters in Protein Crystallization

1. The state and purity of the protein

2. Precipitant (type and concentration)

3. pH - usually test in 1/2 pH units

4. Temperature

5. Protein concentration

Common Precipitants (Salts)

• Salts

ammonium sulfateammonium phosphatesodium/potassium phosphatesodium citratesodium sulfatesodium chloridemagnesium sulfate

Common Precipitants (Organics)

• polymeric precipitants

poly(ethylene glycol) 6000-8000methylether poly(ethylene glycol) 5000poly(ethylene glycol) 1000-2000poly(ethylene glycol) 400

• alcohols

ethanol, propanol2-methyl-2,4,-pentane diol (MPD), propanediol

Vapor Diffusion

• Introduced in the late 1960s

• “Conventional” sitting drop method was introduced by Bob Bock in 1968 (former Dean of the Graduate School at UW)

• Hanging drop method was developed in 1971

• This became the standard method for crystallization by 1972

Hanging Drop Method of Vapor Diffusion

This is the most commonly used technique for surveying and growing crystals for structural analyses.

X-ray Diffraction is the Best Test for Crystallinity

Most protein crystals are grown with the goal of determining their structure, thus an x-ray diffraction experiment is considered to be the ultimate proof that a well ordered crystalline lattice has been achieved.

Common Table Salt

Common Table Sugar

C-3’-Methyltransferase

• Involved in the production of an unusual sugar called tetronitrose

• Tetronitrose is found in an antitumor and antibacterial agent called tetrocarcin A.

C-3’-Methyltransferase

S-Adenosylmethionine

• Discovered in Italy in 1952

• Referred to as SAMe in the vitamin supplement stores

• May be helpful in the treatment of depression, osteoarthritis, fibromyalgia, liver disease, Alzheimer’s Disease

Space-Filling

Ball-and-Stick

Ribbon Representation

Electron Density

His 181

Glu 224

Tyr 222