Crystallography

X-ray Crystallography

Macromolecular X-ray Crystallography

• X-ray crystallography: the technique in the determination of protein structure progressing from the first low-resolution structures of myoglobin to highly refined structures of macromolecular complexes (proteins, viruses, DNA, RNA).

• X-rays - discovered by Willem Röentgen, were shown to be diffracted by crystals in 1912 by Max von Laue.

• Lawrence Bragg, with his father William Bragg, interpreted the patterns of spots obtained on photographic plates located close to crystals exposed to X-rays.

• Bragg’s law: ‘focusing effects’ arise if X-rays are reflected by series of atomic planes and he formulated a direct relationship between the crystal structure and its diffraction pattern.

Electromagnetic Spectrum

Bragg’s Law

• Sets of parallel lattice planes would ‘select’ from incident radiation those wavelengths corresponding to integral multiples of this wavelength.

• Peaks of intensity for the scattered X-rays are observed when the angle of incidence is equal to the angle of scattering and the path length difference is equal to an integer number of wavelengths.

• Bragg’s law allows information about the crystal structure to be determined since the wavelength of X-rays is closely controlled.

• From the Bragg equation, diffraction maxima are observed when the path length difference for the scattered X-rays is a whole number of wavelengths.

The basic crystallography ‘set-up’ used in X-ray diffraction

• Monochromatic X-rays of wavelength 1.5418 A˚, sometimes called Cu Kα X-rays, cause dislodging of an electron from the K shell and the movement of an electron from the next electronic shell (L). After passing through filters to remove Kβ radiation the X-rays strike the crystal

• Detection methods include charged coupled devices (CCD) and are enhanced by synchrotron radiation, an intense source of X-rays.

Protein Production

CrystallisationData Collection Phasing

Protein Structure

DepositionStructure analysis

TargetSelection

Overview of Protein Crystallography

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STRUCTURAL BIOLOGY

PROTEIN CRYSTALLOGRAPHY NMRATOMIC STRUCTURE OF MACROMOLECULESPROTEIN DATA BANK

SYNCHROTRON RADIATION CENTERES

STRUCTURE-BASED DRUG DESIGN

STRUCTURAL GENOMICS CONSORTIUM

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The atomic planes of a crystal cause an incident beam of X-rays (if wavelength is approximately the magnitude of the inter-atomic distance) to interfere with one another as they leave the crystal. The

phenomenon is called X-ray diffraction.

Diffractio

n Pattern of P

rotein Crystal

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MODEL REFINEMENT

ELECTRON DENSITY COMPUTATION

PHASE DETERMINATION

DATA MEASUREMENTS

PROTEIN CRYSTALLIZATION

PROTEIN PURIFICATION

FROM SOLUTION TO STRUCTURE

PROTEIN CRYSTALLOGRAPHY

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PROTEIN PURIFICATION (HPLC…)

From wild type, engineered to overproducing organism

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PROTEIN CRYSTALLIZATION

By de novo crystallization or seeding techniques

Co-crystallization with drug/small molecules

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Sitting Drop Vapor DiffusionHanging Drop Vapor Diffusion

Slow EvaporationDialysis

Common Methods for Crystallization

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Major factors that affect crystallization

1) Purity of proteins

2) Protein concentration

3) Starting conditions (make-up of the protein solution)

4) Precipitating agent (precipitant)

5) Temperature

6) pH

7) Additives: Detergents, reducing agents, substrates, co-factors, etc.

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View the Hampton Protein Crystal Gallery http://www.hamptonresearch.com

Properties of protein crystals

• Soft, easy to crush• Contain large solvent channels

– Relatively large organic and inorganic molecules can diffuse inside

• Anisotropic physical properties– Birefrigence due to anisotropic refraction indices

• Ability to diffract X-ray due to regular spaced lattices

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Protein crystals contain large amounts of solvent

Small molecule crystals are tightly packed and do not

contain solvent

•© 2006

•Academic Press23

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Crystal Mounting

Capillary tubes (Glass or Quartz) Cryo-loops (thin nylon)

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PHASE DETERMINATION

Molecular replacement

Non-crystallographic symmetry or

Direct calculations

Methods based on isoamorphous replacement (preparation of heavy atom derivatives)

Anomalous scattering

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ELECTRON DENSITY MAP COMPUTATION AND INTERPRETATION

Interpretation of mini maps ; Model building on computer graphic displays

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High-throughput Protein crystallography

Structural Genomics

Synchrotron Radiation Centers

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Highthroughput Protein Crystallography Centers:

USA, UK, France, Germany, Canada, Japan, Israel

Joint Center for Structural GenomicsMidwest Center for Structural Genomics

Northeast Structural Genomics ConsortiumNew York SGX Research Center for Structural Genomics

Specialist Centers:Accelerated Technologies Center for Gene to 3D Structure

Center for Eukaryotic Structural Genomics Center for High-Throughput Structural Biology

Center for Structures of Membrane Proteins Integrated Center for Structure and Function Innovation New York Consortium on Membrane Protein Structure

Modeling Centers:Joint Center for Molecular Modeling

New Methods for High-Resolution Comparative Modeling

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Protein crystallography

USA, UK, Italy, France, Germany, Canada, Japan,Singapore, Australia

Armenia, Brazil, China, Denmark, India, Jordan, Netherlands,Russia, South Korea, Spain, Sweden, Switzerland, Taiwan,Thailand

Synchrotron Radiation Centers

Major centers

Small centers

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1. Electron gun 2. Linac 3. Booster ring 4. Storage ring

5. Beamlines 6. Experimental Stations

Electrons are accelerated by the linear accelerator and the booster synchrotron and confined to travel around the storage ring at nearly the speed of light. Accelerating electrons lose energy in the form of synchrotron light.

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DRUG TARGETStructure-Based Drug Design: DRUGGABLE TARGET

Closely linked to human diseaseBinds a small molecule to carry out a function

Well defined binding pocket

Should be essential

Should be unique

should be able to be inhibited by binding a small molecule.