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 Why Crystallography? 

The year 2014 has been declared by the UNESCO - the United Nations Educational, Scientific and Cultural Organization the as the International Year of Crystallography (IYCr), starting January 2014 with an Opening Ceremony at the UNESCO headquarters and world-wide activities in science education. The IYCr therefore ranks prominently in importance amongst other globally relevant themes of UN Observances such as Sustainable Energy, Water Conservation, or Biodiversity. The question is, why should we care about a field of science few of us may ever have heard about?

Let us preface the substantial reasons why in fact a celebration of crystallography is a worthy occasion with two quotes:

"Crystallography borders, naturally, on pure physics, chemistry, biology, mineralogy, technology and also on mathematics, but is distinguished by being concerned with the methods and results of investigating the
arrangement of atoms in matter
, particularly when that arrangement has regular features."
Paul Ewald (1948) Acta Crystallogr. 1, 2

"Almost all aspects of life are engineered at the molecular level, and without understanding molecules we can only have a sketchy understanding of life itself."
Francis Crick, Nobel Laureate and co-discoverer of the DNA structure,  (1988), What Mad Pursuit: A Personal View of Scientific Discovery, p. 61

Molecular structure defines function

The molecular structure of matter defines its properties and function. This simple but far-reaching statement is true for all matter. The properties of gasses, liquids, rocks, minerals, semiconductors, or small organic molecules are defined by their molecular structure, as are the functions of proteins and their complex macromolecular assemblies, the molecular machines of life. The science that allows us to examine matter at the most detailed, atomic or molecular level is crystallography. In all techniques of crystallography the fact is used that atoms or even complex molecules, once arranged in an ordered, periodic pattern - also known as a crystal - diffract X-rays, neutrons, or electrons, which allows to reconstruct the molecular structure in atomic detail. A fundamental requirement for crystallography therefore is the presence of a natural or laboratory-grown crystal of the matter of interest. Although crystallography is in its fundamental principle straightforward (Figure 1), it utilizes some of the most sophisticated machinery at synchrotron facilities, and requires an elaborate reconstruction of the diffraction data into an atomic model of the diffracting matter. It is not just a simple microscopic imaging or viewing technique.

Principle of Crystallography  Figure 1: The principle of X-ray
. A crystal - here consisting of ordered protein molecules - is mounted on a goniostat with at least one rotatable axis. It is exposed to a finely collimated, intense X-ray beam, and diffraction images are recorded on an area detector and combined into a diffraction data set. Diffraction data are transforms of the molecular shape into reciprocal space, equivalent to being transformed into “secret code.” This secret reciprocal space code must be deciphered for each structure by the crystallographer. The basic mathematical tool of back-transformation from reciprocal diffraction space into direct molecular space is the Fourier transform (FT), which together with separately acquired phases for each diffraction spot allows reconstructing the electron density (blue grid) of the molecules self-assembled into the diffracting crystal. An atomic model of the structure, represented in the figure by a ribbon model, is then built into the 3-dimensional electron density. The absence of phases in the diffraction data is the origin of the phase problem in crystallography and a suitable phasing strategy needs to be developed for each structure determination.

The beginnings of X-ray crystallography

Analysis of the first X-ray diffraction patterns, obtained in 1912 by Walther Friedrich and Paul Knipping in Max von Laue’s laboratory (Sidebar 1) from crystals of simple compounds consisting of only one or few different elements such as diamond (figures below), rock salt, or zinc sulfide, confirmed in a fundamental way the atomic constitution of matter and the interactions and bonding of the atoms. Given that the atom theory and particularly quantum mechanics were then in their infancy, these early diffraction experiments provided the much needed crucial evidence and support for the development of the atomic theory of matter (nobody had ever really 'seen' atoms until this point). Interestingly, shortly after X-rays were discovered by Wilhelm Conrad Röntgen in 1895 and even before the first diffraction patterns of simple compounds were recorded, it was already known that also proteins - very complex and large molecules - could form crystals. It would take more than three decades of technical developments until the first diffraction patterns of protein crystals could be recorded and another two decades until the successful determination of the first macromolecular structure, myoglobin, in 1957 (Sidebar 10-1).

Sidebar 1: Early history of Crystallography and its first Nobel prizes. The mathematical formulation of the reflection conditions in a 3-dimensional crystal was formulated first by Max von Laue, who received the Nobel Prize in Physics in 1914 for the discovery of diffraction of X-rays by crystals. His theoretical derivations were tested in 1912 (when he was Privatdozent in Munich) by Arnold Sommerfeld’s assistants W. Friedrich and P. Knipping, who conducted the first diffraction experiments with X-rays. These  experiments nearly did not happen, because Sommerfeld was occupied at this time with the study of the directional dependence of the Bremsstrahlung, and thought little of testing von Laue’s “Raumgitterhypothese.” While the experiments unambiguously established both the electromagnetic wave nature of X-rays and that matter consists of discrete atoms, the three separate diffraction conditions expressed as Laue equations for each direction in the crystal lattice are not overly convenient and not easy to visualize in practical use. Laue’s diffraction pictures were actually obtained using “white” (polychromatic)
X-ray radiation, and were finally interpreted by Sir William Lawrence Bragg. Born in Australia, W. Lawrence Bragg was exposed to X-rays at the early age of 5 years, when he crashed his tricycle and broke his arm. His father W. Henry had recently read about Röntgen’s first X-ray imaging experiments and used the newly discovered X-rays to examine his own son’s broken arm. The affinity to X-rays appears to have stuck with W. Lawrence, and he continued to work in Cambridge on X-ray
diffraction in collaboration with his father, who ran an
X-ray laboratory in Leeds. Sir W. Henry built the first “X-ray
spectrometer,” a prototype of the modern diffractometer
that used monochromatic X-rays, which greatly simplifies
the interpretation of the diffraction patterns. W. Lawrence Bragg finally came up with the brilliant idea to interpret X-ray diffraction as reflection on discrete lattice planes (hkl), leading to the famous and indispensable Bragg
equation that relates the diffraction angle to the lattice spacing dhkl:
Bragg's Equation
When the Braggs received the Nobel Prize in Physics jointly
in 1915
, W. Lawrence was the youngest Nobel recipient ever, at an age of 25 years. An interesting reminiscence of
his early work with W. L. Bragg in Cambridge was compiled
by Austrian-born Nobel laureate Max Perutz, who worked for over three decades in England on the structure of hemoglobin and received the 1962 Nobel Prize in Chemistry together with John Kendrew for their studies of the structures of globular proteins.

Click here for a full account of all Nobel Prizes in or associated with Crystallography.
Crystals - from minerals to therapeutic drugs
Quarz crystal - Wikipedia
A beautiful specimen of a Quartz Scepter Crystal - great beauty but no magic.
Already the simple visual inspection of crystals leads one to suspect that the regularity of faces and dihedral angles between the crystal faces in some form represents the internal order of that crystal. Such ideas have been developed very early on, and the fascinating regularity of crystals strongly influenced the concept of platonic bodies and atomicity during the Greek antique (ca. 400 BC). Even today, in rather enlightened times (at least in secular aspects), the regularity and beauty of crystals has in some circles given rise to the erroneous superstition that crystals harbor special and magic powers. Crystals are certainly special in their properties, but they do not possess any magic powers.

Early attempts of a rational explanation of crystal properties in the 17th century by Huygens and other scientists followed, and a well-developed descriptive crystallography based on careful measurements of mineral crystals emerged. At the end of the 19th century the idea that the macroscopic appearance of crystals is a manifestation of their microscopic regularity was well established, but the final proof had to wait for the actual X-ray diffraction experiments of Max von Laue and Coworkers and by Sir William Henry and his son Sir William Lawrence Bragg (Sidebar 1). From that point on, the tremendous impact of crystallography on the understanding of the physics and chemistry of material properties has led to a quantum leap in our capability to design and engineer the advanced materials we now use on a daily basis. Since the early 1950’s, when protein structure determination became feasible, a similar revolution in our understanding of biological structure and function has taken place, and crystallography plays an indispensable role in modern structure guided drug discovery.

Giant Crystals - Madrid Scientific Film While not all solid matter forms beautiful and well-shaped crystals (some growing to a size of 30 meters in length in subterranean caves, left), much of the solid world is microcrystalline, such as various rocks, composed of billions of tiny microscopic crystals. Some solid materials such as glass are amorphous, that is, they have no precisely ordered internal atomic arrangement.

Why is diamond so hard?

One of the first crystal structures determined by X-ray crystallography was the structure of diamond, a hard crystal created under high pressure and temperature from carbon. The crystal structure immediately explained why diamond is so hard, and why a different crystalline form of carbon, graphite, is soft and can be used as the core of pencils.
Diamond and Graphite - Wikipedia

The diamond structure (bottom left side of Figure 3) shows that each carbon atom is covalently bound to 4 other atoms in a tetrahedral coordination, meaning that very strong atomic binding forces between the atoms extend in 4 directions across the entire crystal - which makes diamond crystals very hard. Compare this to the crystal structure of graphite (right side of Figure 3) - here the carbon atoms are hexagonally arranged in stacked layers. The covalent bonding between the atoms within each layer is still very strong, while the non-bonded interactions between the layers are much weaker. The crystals therefore are soft in one direction with little resistance to sheer - that is why graphite is useful as a pencil core or lubricant. X-ray crystallography has also revealed that for example the color of some gem stone quality diamonds (some are bluish, pink or yellow) originates from substitution of some carbon atoms with nitrogen atoms. Many refractory (hard) materials used in machine cutting tools have also been refined based on the knowledge of atomic substitutions in their material structure. 
C60 Buckyball - Wikipedia Carbon can also form very complex structures such as carbon nanotubes or buckyballs. The structure of those exciting multi-carbon atom nanomaterials useful in applications ranging from electronics to material strengthening and drug delivery was also determined from their crystals by X-ray crystallography. Carbon nanotubes are the strongest and stiffest materials yet discovered in terms of tensile strength and elastic modulus. The Nobel prizes in Chemistry 1996 and in Physics 2010 were awarded for work on complex carbon nanostructures.

 Crystals of the molecules of life

Even the huge protein molecules in the tissues of animals and plants, where they perform a wide variety of functions, are able to self-organize into ordered crystals. Their crystals are at least as beautiful as those of minerals, but there is a crucial difference: They grow only very small, and they are very sensitive and fragile. This can be immediately understood from their crystal structures: there are only few nonbonded contacts connecting huge molecules, with the remaining intermolecular space - on average 50% - filled with the solvent those crystals grew in. One of the biggest surprises from the determination of the first protein crystal structures was the total absence of any symmetry in the protein molecules themselves which made up the beautiful and symmetric protein crystals.
You can watch movies of protein crystals growing (courtesy George Sheldrick and students), crystals absorbing a colored ligand, and get some idea about the process of structure guided drug discovery.

The tetragonal crystals to the right are only 0.3 mm in size (which is actually quite large for protein crystals) and they are viewed through a microscope. They appear blue because they have taken up a colored ligand through their intermolecular solvent channels visible in the packing diagram (far right). Soaking small pharmaceutical molecules in to protein crystals is important for structure-guided therapeutic drug development   Crystals with Ligand4-fold symmetry and solvent channel
Isoniazid boud to INH  How do pharmaceutical drugs work?

X-ray crystal structures can reveal how a small molecule ligand binds to a protein target. As an example, bacterial proteins necessary for the pathogen's function, or protein receptors necessary for the uncontrolled proliferation of a cancer cell can be inhibited by a small molecule therapeutic drug. By examining the binding patterns of the drug or its precursor molecule, we are often able to design a better binding and more specific drug with less side effects

The active form of the antitubercular drug isoniazid

Isoniazid (isonicotinic acid hydrazide) is converted by the mycobacterial enzyme KatG into the active drug isonicotinic acyl-NADH, shown on top as a stick model (yellow: carbon atoms; blue: nitrogen; red: oxygen; purple: phosphorous; hydrogen atoms are usually not seen in X-ray structures and omitted) in its electron density (blue grid, which is what the crystallographer actually sees and interprets) and below the model of the molecule in the binding pocket of the target InhA, an enoyl-acyl-carrier-protein reductase, essential for the biosynthesis of mycolic fatty acids in the bacterium's cell wall. The clear electron density obtained in the 1.75 Å structure of its complex with InhA by Jim Sacchettini confirmed beyond doubt the chemical composition and the inhibiting mode of the modified drug. PDB entry 2nv6.
Human cells do not possess the same fatty-acid rich cell walls as the tuberculosis causing mycobacteria, and therefore the human body does not need an enzyme for the synthesis of this special cell wall component. Humans can safely take this inhibitor and are not affected, while the bacteria die. Sometimes the bacteria can develop resistance to a drug, and a new one must be found, often by modifying the known drugs by analyzing the structures in complex with their protein target.

Keeping your DNA neatly organized.

The human genome is encoded in molecular material called DNA, deoxyribonucleic acid. Four different nucleotides, are connected into long,2-stranded helical DNA macromolecules, consisting in total of approximately 3 billion nucleotide pairs organized into 23 chromosomes. Uncoiled and extended end to end, the DNA would extend about 3 meters, which obviously needs some clever packing to fit into the nucleus of a human cell of about 6 micrometer in diameter. In humans, the genetic material is thus neatly organized in complexes of DNA with core protein histones and other chromosomal proteins that together form nuclear chromatin. The nucleosome core chromatin repeating unit includes two copies each of four core histones H2A, H2B, H3 and H4 (collective molecular mass 206 kDa) wrapped by 146-147 residues of contiguous DNA. The question was, how these nucleosome core particles assemble and how exactly the DNA wraps around the core histone proteins.

Image 2-49  The crystal structure of the nucleosome core particle was the culmination of more than 15 years of work by Timothy Richmond and coworkers involving laborious protein preparation, persistent protein-DNA complex crystallization and solid protein crystallography. The four core histone proteins were individually expressed in milligram quantities in E. coli bacteria, purified under denaturing conditions, then refolded into histone octamers and assembled into nucleosome core particles using 147 base pair defined-sequence DNA fragment derived from human -satellite DNA. The final result was the depicted model containing the entire DNA and histone octamer

The nucleosome core particle
.  The nucleosome core particle consists of two copies each of the four core histone proteins, H2A, H2B, H3 and H4, wrapped by 146 base pairs of DNA. The tails of the histone proteins, some of which are seen protruding from between the base pairs, form strong or weak nucleosomal interactions, depending on their state of acetylation: When hyperacetylated, nucleosomal interactions are weakened, the DNA is not constrained on the surface of the nucleosome and becomes accessible to transcription factors. PDB entry 1aoi.

 Crystallography rocks!

The few selected examples should have demonstrated that crystallography has contributed significantly to much of the progress in all fields of science and engineering which concern themselves with crystallizable matter. The materials with properties understood, improved, and designed based on detailed molecular structure revealed by crystallography range from semiconductors in your cell phone, alloys and advanced materials in aerospace, to the therapeutic drugs you take to maintain your health or to fight disease. All brought to you by crystallography.

Visit here for a quick ttutorial of protein crystallography


If you are really curious about protein drug target crystallography, you can find a full account of the subject in my textbook Biomolecular Crystallography. You can also write me with any comments or questions about crystallography.

Bernhard Rupp
k. k. Hofkristallamt
Ordo Militum Vindicis Crystallographiae
br at hofkristallamt.org

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Production of this IYCr 2014 web page has in part been supported by the European Union under a FP7 Marie Curie People Action, grant PIIF-GA-2011-300025 (SAXCESS). Images courtesy Wikipedia under Creative Commons Attribution-Share Alike 3.0 Unported License or reproduced with permission from Biomolecular Crystallography by Bernhard Rupp, C 2009-2014 Garland Science/Taylor & Francis LLC

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