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Lecture 1

Introduction to X-ray Crystallography

Tuesday, February 1, 2011

Protein Crystallography

Crystal Structure Determination in Principle:From Crystal to Structure

Dr. Susan Yates

Contact Information

Dr. Susan Yates

yates@queensu.ca(please put BCHM313 in subject line)

Botterell Hall, Rm623Office hours by appointment

9 lectures (Feb 1-18, 2011)

Outline – X-ray Crystallography

• Schedule (subject to change)• Lecture 1: Introduction into technique, X-rays,

and Crystals• Lecture 2: Crystals: Theory and Practice • Lecture 3: Instrumentation, Waves and

Diffraction• Lecture 4: Bragg’s law, Handling diffraction data• Lecture 5: Solving the Phase Problem• Lecture 6: Modeling Building, Structure

Refinement• Lecture 7: Accessing Crystal Structure Quality• Lecture 8: The Protein Data Bank, Coordinates• Lecture 9: Crystallography Review

PDB Statistics – Release Entries as of Oct. 19, 2010

0.5%

12.6%

87.0% X-ray Crystallography

NMR Spectroscopy

Electron Microscopy

5959659596 entriesentries

8619 entries8619 entries

(312 entries)(312 entries)

Protein Structure Determination• How?• X-ray crystallography

• NMR spectroscopy

• Electron microscopy

Early Milestones of Crystallography

• 1840• First documented protein crystallization

• Earthworm Hemoglobin by F.L.Hunfield

• 1845• A. Bravais - correctly predicted 14 lattice systems (Later to be called Bravais Lattices)

• 1895• Wilhelm Rontgen observed highly penetrating radiation from fast electrons impinging on matter

• 1912• Max von Laue demonstrated the wave nature of X-rays, by diffraction from a crystal of copper sulphate

• 1913• Sir L. Bragg solved structure of NaCl

Nobel Prize Winners associated with Crystallography

• 1901 Physics (W.C. Röntgen) Discovery of X-rays• 1914 Physics (M. Von Laue) Diffraction of X-rays by crystals• 1915 Physics (W.H. Bragg & W.L. Bragg) Use of X-rays to determine crystal

structure• 1929 Physics (L.-V. de Broglie) The wave nature of the electron• 1937 Physics (C.J. Davisson & G. Thompson) Diffraction of electrons by crystals• 1946 Chemistry (J.B. Sumner) Discovers that enzymes can be crystallized• 1954 Chemistry (L.C. Pauling) Nature of the chemical bond and its application

to the elucidation of the structure of complex substances• 1962 Chemistry (J.C. Kendrew & M. Perutz) For their studies of the structures of

globular proteins• 1962 Physiology/Medicine (F. Crick, J. Watson & M. Wilkins) Helical structure of

DNA• 1964 Chemistry (D. Hodgkin) Structure of many biochemical substances

including Vitamin B12• 1972 Chemistry (C.B. Anfinsen) Folding of protein chains• 1976 Chemistry (W.N. Lipscomb) Structure of boranes• 1982 Physics (K.G. Wilson) Theory of critical phenomena in connection with

phase transitions

Nobel Prize Winners associated with Crystallography

• 1982 Chemistry (A. Klug) Development of crystallographic electron microscopy and discovery of the structure of biologically important nucleic acid–protein complexes

• 1985 Chemistry (H. Hauptman & J. Karle) Development of direct methods for the determination of crystal structures

• 1988 Chemistry (J. Deisenhofer, R. Huber & H. Michel) Determination of the 3-dimensional structure of a photosynthetic reaction centre

• 1991 Physics (P.-G. de Gennes) Methods of discovering order in simple systems can be applied to polymers and liquid crystals

• 1992 Physics (G. Charpak) Discovery of the multi wire proportional chamber• 1994 Physics (C. Shull & N. Brockhouse) Neutron diffraction• 1996 Chemistry (R. Curl, H. Kroto & R. Smalley) Discovery of the fullerene form

of carbon• 1997 Chemistry (P.D. Boyer, J.E. Walker & J.C. Skou) Elucidation of the

enzymatic mechanism underlying the synthesis of ATP and discovery of an ion-transporting enzyme

• 2003 Chemistry (R. MacKinnon) Potassium channels• 2006 Chemistry (R.D. Kornberg) Studies of the molecular basis of eukaryotic

transcription• 2009 Chemistry (V. Ramakrishnan, T.A. Steitz & A.E. Yonath) Studies of the

structure and function of the ribosome

Why is Three-Dimensional Structure Important?

“Picture is worth a thousand words”

What Structure Can Tell You?

• Understand biological processes at the basic level• Understand disease at an atomic level• Help develop new drugs• Engineer new and improved proteins for various applications

• Protein fold• Infer sequence-structure relationships• Enzyme mechanisms (to some extent)• Protein-protein, -DNA, -RNA interfaces• Ligand-binding sites• Conformational changes• Flexible regions

Looking at the Objects Around You

• Use your eyes/microscope/telescope!

• Solving a structure purely “observationally”• Works for cells, tissues, galaxies etc.• Look at the structure and describe or modelwhat you see

• So why can’t we just look at a protein withmicroscope?

Because…• Even a light microscope has its limits

• “Diffraction limit“• Cannot image things that are much smaller than the wavelength of the light you are using

• Wavelength for visible light (400-700 nm) but atoms are separated by distances of the order of 0.1 nm (1 Å)

Diffraction Limit• Fundamental physical principle• In order to see a detail (x) meters in extent, the illuminating radiation you use must have a wavelength at most double that size

• So… protein molecules are invisible to visiblelight!• Even a concentrated solution of protein istransparent

Light passes without interacting, so noinformation on the protein is encoded inthe emission

How about X-rays?

X-rays!

Wavelength range from 0.1-100 Å

10-8 cm = 1 Å

Picking your “Ruler”• To measure something accurately, you need the appropriate ruler• Distance between cities, use kilometres• Length of your hand, use centimetres

• Crystallographers measure distances between atoms in Å

• Perfect "rulers" to measure Å distancesare X-rays• X-rays used by crystallographers are ~ 0.5 to 1.5 Å• Just the right size to measure the distance between atoms in a molecule

X-rays!• They are close to inter-atomic distance!

• To measure atomic distances through interference and to determine the structure of molecules, our “ruler” must have atomic dimensions

• This is why X-rays are required for crystallographic structure determinations

How Does a Microscope Work?

• Light strikes the object and is diffracted in various directions

• The lens collects the diffracted rays and reassembles them to form an image

Lens focuses visible light

An X-ray Microscope?• Can't build an X-ray microscope • No X-ray lens

• With X-rays, we can detect diffraction from molecules, but we need a different approach to reassemble the image

Lens focuses visible light, but the refractive index for very short

wavelengths is ~ 1, so far no material can be used to focus X-rays

Let’s Begin our Journey…

X-RAYCRYSTALLOGRAPHY

Microscope vs. X-ray CrystallographyOptical microscope

"Impossible" X-ray microscope

X-ray Crystallography• High-powered X-rays are aimed at a tiny crystal containing trillions of identical molecules

• Crystal scatters the X-rays onto an electronic detector • Like a disco ball spraying light across a dance floor

• Electronic detector similar to those used to capture images in a digital camera

Steps in Solving an X-ray Structure

Know Your Protein• Sequence, molecular weight• Disulfides, glycoprotein?, phosphorylated?• Maximum stability/activity, degradation?• Cofactors• Tags used in purification• Secondary structure prediction• Homologs with known structure

A Good Protein Sample• Pure• SDS-PAGE, Mono Q, IEF, mass spectroscopy

• Defined buffer• Defined concentration• No aggregation• Dynamic light scattering, size exclusion• To improve: salt, pH, temperature, detergent, batch, cofactors, binding partners, mutagenesis

• Need lots of protein too!

Steps in Solving an X-ray Structure

Why Crystals?

• X-ray scattering from a single molecule would be unimaginably weak and could never be detected above the noise level (scattering from air and water)

• A crystal arranges huge numbers of molecules in the same orientation, so that scattered waves can add up in phase and raise the signal to a measurable level

• A crystal acts as an amplifier!

What is a Crystal?

Crystal acts as an X-ray diffraction amplifier

Packing of Molecule

Three-Dimensional Crystals• Periodic array of atoms, molecules, viruses...• Translational symmetry along three vectors a, b, c

Crystal Lattice• Periodic arrangement in 3 dimensions• A crystal unit cell is defined by its cell constants and is the building block for the whole crystal• Edges: a, b, c• Angles: α, β, γ

Next time…• Building crystals• Theory and Practice