structure of proteins by x-ray crystallography

Structure of proteins by X-ray crystallography

Wei Huang Spring 2011

Crystal Growth

•  Sitting Drop Vapor Diffusion

•  For more crystal growth techniques, go to: http://hamptonresearch.com/growth_101_lit.aspx

Supersaturation •  Supersaturation can be achieved by adding more of a substance (to a

solution) than can normally be dissolved. This is a thermodynamically unstable state, achieved most often in protein crystallography by vapor diffusion or other slow evaporation techniques.

Zone 1 - Metastable zone. The solution may not nucleate for a long time but this zone will sustain growth. It is frequently necessary to add a seed crystal. Zone 2 - Nucleation zone. Protein crystals nucleate and grow. Zone 3 - Precipitation zone. Proteins do not nucleate but precipitate out of solution.

X-ray source

CAMD synchrotron

In-house X-ray source

Next lab (Thursday): a tour to visit some facilities for X-ray crystallography on the 5th floor in Choppin Hall. Meet in front of Room 527.

•  X-ray crystallography = microscope without

lenses & optical λ ~1.5 Å •  Bond lengths ~ 1.4 Å •  Electrons scatter X-rays

X-rays “see electrons” •  Resolution—Best is λ/2 Typical is 1 to 3 Å

Determining Atomic Structure

dmin =!

2sin"max

X-ray diffraction

Structural Refinement

crystal

Intensities I(h,k,l)

Experimental •  Heavy atom labels •  Selenium for sulfur •  Trial & error similar structure

Phases

Electron density ρ(x,y,z)

Atom positions (x,y,z)

Known: Amino acid sequences Ligands Bond lengths angles Constraints on geometry

Fourier Transform

Introduction to DeepView (Swiss-PdbViewer) & Exercise

DeepView: Main controls

DeepView: Control Panel

Exercise 1

•  2XBR.pdb contains at least one occurrence each of 20 different amino acid residues. Use this file to become familiar with the basic 20 residues. Locate each of the 20 amino acid residues, memorize its 3D structure. You should be able to recognize it as well as draw it. You may be tempted to rush through this part of the exercise, but if you take time now to commit each structure to memory, the assignment below will be much easier to complete and you will be almost ready for this part in the practical exam.