HOME
The Info List - X-ray Diffraction


--- Advertisement ---



(i) (i) (i) (i) (i)

X-RAY CRYSTALLOGRAPHY is a technique used for determining the atomic and molecular structure of a crystal , in which the crystalline atoms cause a beam of incident X-rays
X-rays
to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density , the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds , their disorder , and various other information.

Since many materials can form crystals—such as salts , metals , minerals , semiconductors , as well as various inorganic, organic, and biological molecules— X-ray
X-ray
crystallography has been fundamental in the development of many scientific fields. In its first decades of use, this method determined the size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences among various materials, especially minerals and alloys . The method also revealed the structure and function of many biological molecules, including vitamins , drugs, proteins and nucleic acids such as DNA
DNA
. X-ray crystallography is still the chief method for characterizing the atomic structure of new materials and in discerning materials that appear similar by other experiments . X-ray
X-ray
crystal structures can also account for unusual electronic or elastic properties of a material, shed light on chemical interactions and processes, or serve as the basis for designing pharmaceuticals against diseases .

In a single-crystal X-ray diffraction
X-ray diffraction
measurement, a crystal is mounted on a goniometer . The goniometer is used to position the crystal at selected orientations. The crystal is illuminated with a finely focused monochromatic beam of X-rays, producing a diffraction pattern of regularly spaced spots known as reflections. The two-dimensional images taken at different orientations are converted into a three-dimensional model of the density of electrons within the crystal using the mathematical method of Fourier transforms , combined with chemical data known for the sample. Poor resolution (fuzziness) or even errors may result if the crystals are too small, or not uniform enough in their internal makeup.

X-ray
X-ray
crystallography is related to several other methods for determining atomic structures. Similar diffraction patterns can be produced by scattering electrons or neutrons , which are likewise interpreted by Fourier transformation . If single crystals of sufficient size cannot be obtained, various other X-ray
X-ray
methods can be applied to obtain less detailed information; such methods include fiber diffraction , powder diffraction and (if the sample is not crystallized) small-angle X-ray
X-ray
scattering (SAXS). If the material under investigation is only available in the form of nanocrystalline powders or suffers from poor crystallinity, the methods of electron crystallography can be applied for determining the atomic structure.

For all above mentioned X-ray diffraction
X-ray diffraction
methods, the scattering is elastic ; the scattered X-rays
X-rays
have the same wavelength as the incoming X-ray. By contrast, inelastic X-ray
X-ray
scattering methods are useful in studying excitations of the sample, rather than the distribution of its atoms.

CONTENTS

* 1 History

* 1.1 Early scientific history of crystals and X-rays
X-rays
* 1.2 X-ray diffraction
X-ray diffraction
* 1.3 Scattering * 1.4 Development from 1912 to 1920 * 1.5 Cultural and aesthetic importance

* 2 Contributions to chemistry and material science

* 2.1 Mineralogy
Mineralogy
and metallurgy * 2.2 Early organic and small biological molecules * 2.3 Biological macromolecular crystallography

* 3 Relationship to other scattering techniques

* 3.1 Elastic vs. inelastic scattering * 3.2 Other X-ray
X-ray
techniques * 3.3 Electron
Electron
and neutron diffraction

* 4 Methods

* 4.1 Overview of single-crystal X-ray diffraction
X-ray diffraction

* 4.1.1 Procedure * 4.1.2 Limitations

* 4.2 Crystallization
Crystallization

* 4.3 Data collection

* 4.3.1 Mounting the crystal

* 4.3.2 X-ray
X-ray
sources

* 4.3.2.1 Rotating anode * 4.3.2.2 Synchrotron radiation * 4.3.2.3 Free electron laser
Free electron laser

* 4.3.3 Recording the reflections

* 4.4 Data analysis

* 4.4.1 Crystal
Crystal
symmetry, unit cell, and image scaling * 4.4.2 Initial phasing * 4.4.3 Model building and phase refinement * 4.4.4 Disorder

* 4.5 Deposition of the structure

* 5 Diffraction
Diffraction
theory

* 5.1 Intuitive understanding by Bragg\'s law * 5.2 Scattering as a Fourier transform
Fourier transform
* 5.3 Friedel and Bijvoet mates * 5.4 Ewald\'s sphere * 5.5 Patterson function * 5.6 Advantages of a crystal

* 6 Nobel Prizes involving X-ray
X-ray
crystallography

* 7 Applications of X-ray diffraction
X-ray diffraction

* 7.1 X-ray
X-ray
method for investigation of drugs * 7.2 X-ray
X-ray
method for investigation of textile fibers and polymers * 7.3 X-ray
X-ray
method for investigation of bones

* 8 See also * 9 References

* 10 Further reading

* 10.1 International Tables for Crystallography
Crystallography
* 10.2 Bound collections of articles * 10.3 Textbooks * 10.4 Applied computational data analysis * 10.5 Historical

* 11 External links

* 11.1 Tutorials * 11.2 Primary databases * 11.3 Derivative databases * 11.4 Structural validation

HISTORY

EARLY SCIENTIFIC HISTORY OF CRYSTALS AND X-RAYS

Drawing of square (Figure A, above) and hexagonal (Figure B, below) packing from Kepler\'s work, Strena seu de Nive Sexangula.

Crystals, though long admired for their regularity and symmetry, were not investigated scientifically until the 17th century. Johannes Kepler hypothesized in his work Strena seu de Nive Sexangula (A New Year's Gift of Hexagonal Snow) (1611) that the hexagonal symmetry of snowflake crystals was due to a regular packing of spherical water particles. As shown by X-ray
X-ray
crystallography, the hexagonal symmetry of snowflakes results from the tetrahedral arrangement of hydrogen bonds about each water molecule. The water molecules are arranged similarly to the silicon atoms in the tridymite polymorph of SiO2. The resulting crystal structure has hexagonal symmetry when viewed along a principal axis.

The Danish scientist Nicolas Steno (1669) pioneered experimental investigations of crystal symmetry. Steno showed that the angles between the faces are the same in every exemplar of a particular type of crystal, and René Just Haüy
René Just Haüy
(1784) discovered that every face of a crystal can be described by simple stacking patterns of blocks of the same shape and size. Hence, William Hallowes Miller in 1839 was able to give each face a unique label of three small integers, the Miller indices which remain in use today for identifying crystal faces. Haüy's study led to the correct idea that crystals are a regular three-dimensional array (a Bravais lattice ) of atoms and molecules ; a single unit cell is repeated indefinitely along three principal directions that are not necessarily perpendicular. In the 19th century, a complete catalog of the possible symmetries of a crystal was worked out by Johan Hessel , Auguste Bravais , Evgraf Fedorov , Arthur Schönflies and (belatedly) William Barlow (1894). From the available data and physical reasoning, Barlow proposed several crystal structures in the 1880s that were validated later by X-ray
X-ray
crystallography; however, the available data were too scarce in the 1880s to accept his models as conclusive. X-ray crystallography shows the arrangement of water molecules in ice, revealing the hydrogen bonds (1) that hold the solid together. Few other methods can determine the structure of matter with such precision (resolution).

Wilhelm Röntgen discovered X-rays
X-rays
in 1895, just as the studies of crystal symmetry were being concluded. Physicists were initially uncertain of the nature of X-rays, but soon suspected (correctly) that they were waves of electromagnetic radiation , in other words, another form of light . At that time, the wave model of light —specifically, the Maxwell theory of electromagnetic radiation —was well accepted among scientists, and experiments by Charles Glover Barkla showed that X-rays
X-rays
exhibited phenomena associated with electromagnetic waves, including transverse polarization and spectral lines akin to those observed in the visible wavelengths. Single-slit experiments in the laboratory of Arnold Sommerfeld suggested that X-rays
X-rays
had a wavelength of about 1 angstrom . However, X-rays
X-rays
are composed of photons , and thus are not only waves of electromagnetic radiation but also exhibit particle-like properties. Albert Einstein
Albert Einstein
introduced the photon concept in 1905, but it was not broadly accepted until 1922, when Arthur Compton
Arthur Compton
confirmed it by the scattering of X-rays
X-rays
from electrons. Therefore, these particle-like properties of X-rays, such as their ionization of gases, caused William Henry Bragg
William Henry Bragg
to argue in 1907 that X-rays
X-rays
were not electromagnetic radiation. Nevertheless, Bragg's view was not broadly accepted and the observation of X-ray diffraction by Max von Laue
Max von Laue
in 1912 confirmed for most scientists that X-rays
X-rays
were a form of electromagnetic radiation.

X-RAY DIFFRACTION

The incoming beam (coming from upper left) causes each scatterer to re-radiate a small portion of its intensity as a spherical wave. If scatterers are arranged symmetrically with a separation d, these spherical waves will be in sync (add constructively) only in directions where their path-length difference 2d sin θ equals an integer multiple of the wavelength λ. In that case, part of the incoming beam is deflected by an angle 2θ, producing a reflection spot in the diffraction pattern .

Crystals are regular arrays of atoms, and X-rays
X-rays
can be considered waves of electromagnetic radiation. Atoms scatter X-ray
X-ray
waves, primarily through the atoms' electrons. Just as an ocean wave striking a lighthouse produces secondary circular waves emanating from the lighthouse, so an X-ray
X-ray
striking an electron produces secondary spherical waves emanating from the electron. This phenomenon is known as elastic scattering , and the electron (or lighthouse) is known as the scatterer. A regular array of scatterers produces a regular array of spherical waves. Although these waves cancel one another out in most directions through destructive interference , they add constructively in a few specific directions, determined by Bragg\'s law : 2 d sin = n {displaystyle 2dsin theta =nlambda }

Here d is the spacing between diffracting planes, {displaystyle theta } is the incident angle, n is any integer, and λ is the wavelength of the beam. These specific directions appear as spots on the diffraction pattern called reflections. Thus, X-ray diffraction results from an electromagnetic wave (the X-ray) impinging on a regular array of scatterers (the repeating arrangement of atoms within the crystal).

X-rays
X-rays
are used to produce the diffraction pattern because their wavelength λ is typically the same order of magnitude (1–100 angstroms) as the spacing d between planes in the crystal. In principle, any wave impinging on a regular array of scatterers produces diffraction , as predicted first by Francesco Maria Grimaldi in 1665. To produce significant diffraction, the spacing between the scatterers and the wavelength of the impinging wave should be similar in size. For illustration, the diffraction of sunlight through a bird's feather was first reported by James Gregory in the later 17th century. The first artificial diffraction gratings for visible light were constructed by David Rittenhouse in 1787, and Joseph von Fraunhofer in 1821. However, visible light has too long a wavelength (typically, 5500 angstroms) to observe diffraction from crystals. Prior to the first X-ray diffraction
X-ray diffraction
experiments, the spacings between lattice planes in a crystal were not known with certainty.

The idea that crystals could be used as a diffraction grating for X-rays
X-rays
arose in 1912 in a conversation between Paul Peter Ewald and Max von Laue
Max von Laue
in the English Garden in Munich
Munich
. Ewald had proposed a resonator model of crystals for his thesis, but this model could not be validated using visible light , since the wavelength was much larger than the spacing between the resonators. Von Laue realized that electromagnetic radiation of a shorter wavelength was needed to observe such small spacings, and suggested that X-rays
X-rays
might have a wavelength comparable to the unit-cell spacing in crystals. Von Laue worked with two technicians, Walter Friedrich and his assistant Paul Knipping, to shine a beam of X-rays
X-rays
through a copper sulfate crystal and record its diffraction on a photographic plate . After being developed, the plate showed a large number of well-defined spots arranged in a pattern of intersecting circles around the spot produced by the central beam. Von Laue developed a law that connects the scattering angles and the size and orientation of the unit-cell spacings in the crystal, for which he was awarded the Nobel Prize in Physics in 1914.

SCATTERING

As described in the mathematical derivation below , the X-ray scattering is determined by the density of electrons within the crystal. Since the energy of an X-ray
X-ray
is much greater than that of a valence electron, the scattering may be modeled as Thomson scattering , the interaction of an electromagnetic ray with a free electron. This model is generally adopted to describe the polarization of the scattered radiation.

The intensity of Thomson scattering for one particle with mass m and charge q is: I o = I e ( q 4 m 2 c 4 ) 1 + cos 2 2 2 = I e 7.94.10 26 1 + cos 2 2 2 = I e f {displaystyle I_{o}=I_{e}left({frac {q^{4}}{m^{2}c^{4}}}right){frac {1+cos ^{2}2theta }{2}}=I_{e}7.94.10^{-26}{frac {1+cos ^{2}2theta }{2}}=I_{e}f}

Hence the atomic nuclei, which are much heavier than an electron, contribute negligibly to the scattered X-rays.

DEVELOPMENT FROM 1912 TO 1920

Although diamonds (top left) and graphite (top right) are identical in chemical composition—being both pure carbon —X-ray crystallography revealed the arrangement of their atoms (bottom) accounts for their different properties. In diamond, the carbon atoms are arranged tetrahedrally and held together by single covalent bonds , making it strong in all directions. By contrast, graphite is composed of stacked sheets. Within the sheet, the bonding is covalent and has hexagonal symmetry, but there are no covalent bonds between the sheets, making graphite easy to cleave into flakes.

After Von Laue's pioneering research, the field developed rapidly, most notably by physicists William Lawrence Bragg
William Lawrence Bragg
and his father William Henry Bragg
William Henry Bragg
. In 1912–1913, the younger Bragg developed Bragg\'s law , which connects the observed scattering with reflections from evenly spaced planes within the crystal. The Braggs, father and son, shared the 1915 Nobel Prize in Physics
Nobel Prize in Physics
for their work in crystallography. The earliest structures were generally simple and marked by one-dimensional symmetry. However, as computational and experimental methods improved over the next decades, it became feasible to deduce reliable atomic positions for more complicated two- and three-dimensional arrangements of atoms in the unit-cell.

The potential of X-ray
X-ray
crystallography for determining the structure of molecules and minerals—then only known vaguely from chemical and hydrodynamic experiments—was realized immediately. The earliest structures were simple inorganic crystals and minerals, but even these revealed fundamental laws of physics and chemistry. The first atomic-resolution structure to be "solved" (i.e., determined) in 1914 was that of table salt . The distribution of electrons in the table-salt structure showed that crystals are not necessarily composed of covalently bonded molecules, and proved the existence of ionic compounds . The structure of diamond was solved in the same year, proving the tetrahedral arrangement of its chemical bonds and showing that the length of C–C single bond was 1.52 angstroms. Other early structures included copper , calcium fluoride (CaF2, also known as fluorite), calcite (CaCO3) and pyrite (FeS2) in 1914; spinel (MgAl2O4) in 1915; the rutile and anatase forms of titanium dioxide (TiO2) in 1916; pyrochroite Mn(OH)2 and, by extension, brucite Mg(OH)2 in 1919;. Also in 1919 sodium nitrate (NaNO3) and caesium dichloroiodide (CsICl2) were determined by Ralph Walter Graystone Wyckoff , and the wurtzite (hexagonal ZnS) structure became known in 1920.

The structure of graphite was solved in 1916 by the related method of powder diffraction , which was developed by Peter Debye
Peter Debye
and Paul Scherrer and, independently, by Albert Hull in 1917. The structure of graphite was determined from single-crystal diffraction in 1924 by two groups independently. Hull also used the powder method to determine the structures of various metals, such as iron and magnesium.

CULTURAL AND AESTHETIC IMPORTANCE

In what has been called his scientific autobiography, The Development of X-ray
X-ray
Analysis, Sir William Lawrence Bragg
William Lawrence Bragg
mentioned that he believed the field of crystallography was particularly welcoming to women because the techno-aesthetics of the molecular structures resembled textiles and household objects. Bragg was known to compare crystal formation to "curtains, wallpapers, mosaics, and roses".

In 1951, the Festival Pattern Group at the Festival of Britain
Festival of Britain
hosted a collaborative group of textile manufacturers and experienced crystallographers to design lace and prints based on the X-ray crystallography of insulin , china clay , and hemoglobin . One of the leading scientists of the project was Dr. Helen Megaw (1907–2002), the Assistant Director of Research at the Cavendish Laboratory in Cambridge at the time. Megaw is credited as one of the central figures who took inspiration from crystal diagrams and saw their potential in design. In 2008, the Wellcome Collection in London curated an exhibition on the Festival Pattern Group called "From Atom
Atom
to Patterns".

CONTRIBUTIONS TO CHEMISTRY AND MATERIAL SCIENCE

X-ray
X-ray
crystallography has led to a better understanding of chemical bonds and non-covalent interactions . The initial studies revealed the typical radii of atoms, and confirmed many theoretical models of chemical bonding, such as the tetrahedral bonding of carbon in the diamond structure, the octahedral bonding of metals observed in ammonium hexachloroplatinate (IV), and the resonance observed in the planar carbonate group and in aromatic molecules. Kathleen Lonsdale 's 1928 structure of hexamethylbenzene established the hexagonal symmetry of benzene and showed a clear difference in bond length between the aliphatic C–C bonds and aromatic C–C bonds; this finding led to the idea of resonance between chemical bonds, which had profound consequences for the development of chemistry. Her conclusions were anticipated by William Henry Bragg
William Henry Bragg
, who published models of naphthalene and anthracene in 1921 based on other molecules, an early form of molecular replacement .

Also in the 1920s, Victor Moritz Goldschmidt and later Linus Pauling developed rules for eliminating chemically unlikely structures and for determining the relative sizes of atoms. These rules led to the structure of brookite (1928) and an understanding of the relative stability of the rutile , brookite and anatase forms of titanium dioxide .

The distance between two bonded atoms is a sensitive measure of the bond strength and its bond order ; thus, X-ray
X-ray
crystallographic studies have led to the discovery of even more exotic types of bonding in inorganic chemistry , such as metal-metal double bonds, metal-metal quadruple bonds, and three-center, two-electron bonds. X-ray
X-ray
crystallography—or, strictly speaking, an inelastic Compton scattering experiment—has also provided evidence for the partly covalent character of hydrogen bonds . In the field of organometallic chemistry , the X-ray
X-ray
structure of ferrocene initiated scientific studies of sandwich compounds , while that of Zeise\'s salt stimulated research into "back bonding" and metal-pi complexes. Finally, X-ray
X-ray
crystallography had a pioneering role in the development of supramolecular chemistry , particularly in clarifying the structures of the crown ethers and the principles of host-guest chemistry .

In material sciences, many complicated inorganic and organometallic systems have been analyzed using single-crystal methods, such as fullerenes , metalloporphyrins , and other complicated compounds. Single-crystal diffraction is also used in the pharmaceutical industry , due to recent problems with polymorphs . The major factors affecting the quality of single-crystal structures are the crystal's size and regularity; recrystallization is a commonly used technique to improve these factors in small-molecule crystals. The Cambridge Structural Database contains over 800,000 structures as of September 2016; over 99% of these structures were determined by X-ray
X-ray
diffraction.

MINERALOGY AND METALLURGY

First X-ray diffraction
X-ray diffraction
view of Martian soil CheMin analysis reveals feldspar , pyroxenes , olivine and more ( Curiosity rover at "Rocknest ", October 17, 2012).

Since the 1920s, X-ray diffraction
X-ray diffraction
has been the principal method for determining the arrangement of atoms in minerals and metals . The application of X-ray
X-ray
crystallography to mineralogy began with the structure of garnet , which was determined in 1924 by Menzer. A systematic X-ray
X-ray
crystallographic study of the silicates was undertaken in the 1920s. This study showed that, as the Si /O ratio is altered, the silicate crystals exhibit significant changes in their atomic arrangements. Machatschki extended these insights to minerals in which aluminium substitutes for the silicon atoms of the silicates. The first application of X-ray
X-ray
crystallography to metallurgy likewise occurred in the mid-1920s. Most notably, Linus Pauling 's structure of the alloy Mg2Sn led to his theory of the stability and structure of complex ionic crystals.

On October 17, 2012, the Curiosity rover on the planet Mars
Mars
at "Rocknest " performed the first X-ray diffraction
X-ray diffraction
analysis of Martian soil . The results from the rover's CheMin analyzer revealed the presence of several minerals, including feldspar , pyroxenes and olivine , and suggested that the Martian soil in the sample was similar to the "weathered basaltic soils " of Hawaiian volcanoes .

EARLY ORGANIC AND SMALL BIOLOGICAL MOLECULES

The three-dimensional structure of penicillin , solved by Dorothy Crowfoot Hodgkin in 1945. The green, white, red, yellow and blue spheres represent atoms of carbon , hydrogen , oxygen , sulfur and nitrogen , respectively.

The first structure of an organic compound, hexamethylenetetramine , was solved in 1923. This was followed by several studies of long-chain fatty acids , which are an important component of biological membranes. In the 1930s, the structures of much larger molecules with two-dimensional complexity began to be solved. A significant advance was the structure of phthalocyanine , a large planar molecule that is closely related to porphyrin molecules important in biology, such as heme , corrin and chlorophyll .

X-ray
X-ray
crystallography of biological molecules took off with Dorothy Crowfoot Hodgkin , who solved the structures of cholesterol (1937), penicillin (1946) and vitamin B12 (1956), for which she was awarded the Nobel Prize in Chemistry
Nobel Prize in Chemistry
in 1964. In 1969, she succeeded in solving the structure of insulin , on which she worked for over thirty years. Ribbon diagram of the structure of myoglobin , showing colored alpha helices . Such proteins are long, linear molecules with thousands of atoms; yet the relative position of each atom has been determined with sub-atomic resolution by X-ray
X-ray
crystallography. Since it is difficult to visualize all the atoms at once, the ribbon shows the rough path of the protein polymer from its N-terminus (blue) to its C-terminus (red).

BIOLOGICAL MACROMOLECULAR CRYSTALLOGRAPHY

Crystal
Crystal
structures of proteins (which are irregular and hundreds of times larger than cholesterol) began to be solved in the late 1950s, beginning with the structure of sperm whale myoglobin by Sir John Cowdery Kendrew , for which he shared the Nobel Prize in Chemistry with Max Perutz
Max Perutz
in 1962. Since that success, 132055 X-ray
X-ray
crystal structures of proteins, nucleic acids and other biological molecules have been determined. For comparison, the nearest competing method in terms of structures analyzed is nuclear magnetic resonance (NMR) spectroscopy , which has resolved 11904 chemical structures. Moreover, crystallography can solve structures of arbitrarily large molecules, whereas solution-state NMR is restricted to relatively small ones (less than 70 kDa ). X-ray
X-ray
crystallography is now used routinely by scientists to determine how a pharmaceutical drug interacts with its protein target and what changes might improve it. However, intrinsic membrane proteins remain challenging to crystallize because they require detergents or other means to solubilize them in isolation, and such detergents often interfere with crystallization. Such membrane proteins are a large component of the genome, and include many proteins of great physiological importance, such as ion channels and receptors . Helium cryogenics are used to prevent radiation damage in protein crystals.

On the other end of the size scale, even relatively small molecules may pose challenges for the resolving power of X-ray
X-ray
crystallography. The structure assigned in 1991 to the antibiotic isolated from a marine organism, diazonamide A (C40H34Cl2N6O6, molar mass 765.65 g/mol), proved to be incorrect by the classical proof of structure: a synthetic sample was not identical to the natural product. The mistake was attributed to the inability of X-ray
X-ray
crystallography to distinguish between the correct -OH / >NH and the interchanged -NH2 / -O- groups in the incorrect structure. With advances in instrumentation, however, it is now routinely possible to distinguish between such similar groups using modern single-crystal X-ray diffractometers.

RELATIONSHIP TO OTHER SCATTERING TECHNIQUES

Further information: X-ray
X-ray
scattering techniques

ELASTIC VS. INELASTIC SCATTERING

X-ray
X-ray
crystallography is a form of elastic scattering ; the outgoing X-rays
X-rays
have the same energy, and thus same wavelength, as the incoming X-rays, only with altered direction. By contrast, inelastic scattering occurs when energy is transferred from the incoming X-ray
X-ray
to the crystal, e.g., by exciting an inner-shell electron to a higher energy level . Such inelastic scattering reduces the energy (or increases the wavelength) of the outgoing beam. Inelastic scattering is useful for probing such excitations of matter, but not in determining the distribution of scatterers within the matter, which is the goal of X-ray
X-ray
crystallography.

X-rays
X-rays
range in wavelength from 10 to 0.01 nanometers ; a typical wavelength used for crystallography is 1 Å (0.1 nm), which is on the scale of covalent chemical bonds and the radius of a single atom. Longer-wavelength photons (such as ultraviolet radiation ) would not have sufficient resolution to determine the atomic positions. At the other extreme, shorter-wavelength photons such as gamma rays are difficult to produce in large numbers, difficult to focus, and interact too strongly with matter, producing particle-antiparticle pairs . Therefore, X-rays
X-rays
are the "sweetspot" for wavelength when determining atomic-resolution structures from the scattering of electromagnetic radiation .

OTHER X-RAY TECHNIQUES

Other forms of elastic X-ray
X-ray
scattering include powder diffraction , Small-Angle X-ray
X-ray
Scattering ( SAXS ) and several types of X-ray
X-ray
fiber diffraction , which was used by Rosalind Franklin
Rosalind Franklin
in determining the double-helix structure of DNA
DNA
. In general, single-crystal X-ray diffraction offers more structural information than these other techniques; however, it requires a sufficiently large and regular crystal, which is not always available.

These scattering methods generally use monochromatic X-rays, which are restricted to a single wavelength with minor deviations. A broad spectrum of X-rays
X-rays
(that is, a blend of X-rays
X-rays
with different wavelengths) can also be used to carry out X-ray
X-ray
diffraction, a technique known as the Laue method. This is the method used in the original discovery of X-ray
X-ray
diffraction. Laue scattering provides much structural information with only a short exposure to the X-ray
X-ray
beam, and is therefore used in structural studies of very rapid events (Time resolved crystallography ). However, it is not as well-suited as monochromatic scattering for determining the full atomic structure of a crystal and therefore works better with crystals with relatively simple atomic arrangements.

The Laue back reflection mode records X-rays
X-rays
scattered backwards from a broad spectrum source. This is useful if the sample is too thick for X-rays
X-rays
to transmit through it. The diffracting planes in the crystal are determined by knowing that the normal to the diffracting plane bisects the angle between the incident beam and the diffracted beam. A Greninger chart can be used to interpret the back reflection Laue photograph.

ELECTRON AND NEUTRON DIFFRACTION

Other particles, such as electrons and neutrons , may be used to produce a diffraction pattern . Although electron, neutron, and X-ray scattering are based on different physical processes, the resulting diffraction patterns are analyzed using the same coherent diffraction imaging techniques.

As derived below, the electron density within the crystal and the diffraction patterns are related by a simple mathematical method, the Fourier transform
Fourier transform
, which allows the density to be calculated relatively easily from the patterns. However, this works only if the scattering is weak, i.e., if the scattered beams are much less intense than the incoming beam. Weakly scattered beams pass through the remainder of the crystal without undergoing a second scattering event. Such re-scattered waves are called "secondary scattering" and hinder the analysis. Any sufficiently thick crystal will produce secondary scattering, but since X-rays
X-rays
interact relatively weakly with the electrons, this is generally not a significant concern. By contrast, electron beams may produce strong secondary scattering even for relatively thin crystals (>100 nm). Since this thickness corresponds to the diameter of many viruses , a promising direction is the electron diffraction of isolated macromolecular assemblies , such as viral capsids and molecular machines, which may be carried out with a cryo-electron microscope . Moreover, the strong interaction of electrons with matter (about 1000 times stronger than for X-rays) allows determination of the atomic structure of extremely small volumes. The field of applications for electron crystallography ranges from bio molecules like membrane proteins over organic thin films to the complex structures of (nanocrystalline) intermetallic compounds and zeolites.

Neutron
Neutron
diffraction is an excellent method for structure determination, although it has been difficult to obtain intense, monochromatic beams of neutrons in sufficient quantities. Traditionally, nuclear reactors have been used, although sources producing neutrons by spallation are becoming increasingly available. Being uncharged, neutrons scatter much more readily from the atomic nuclei rather than from the electrons. Therefore, neutron scattering is very useful for observing the positions of light atoms with few electrons, especially hydrogen , which is essentially invisible in the X-ray
X-ray
diffraction. Neutron
Neutron
scattering also has the remarkable property that the solvent can be made invisible by adjusting the ratio of normal water , H2O, and heavy water , D2O.

METHODS

OVERVIEW OF SINGLE-CRYSTAL X-RAY DIFFRACTION

Workflow for solving the structure of a molecule by X-ray crystallography.

The oldest and most precise method of X-ray
X-ray
crystallography is single-crystal X-ray
X-ray
diffraction, in which a beam of X-rays
X-rays
strikes a single crystal, producing scattered beams. When they land on a piece of film or other detector, these beams make a diffraction pattern of spots; the strengths and angles of these beams are recorded as the crystal is gradually rotated. Each spot is called a reflection, since it corresponds to the reflection of the X-rays
X-rays
from one set of evenly spaced planes within the crystal. For single crystals of sufficient purity and regularity, X-ray diffraction
X-ray diffraction
data can determine the mean chemical bond lengths and angles to within a few thousandths of an angstrom and to within a few tenths of a degree , respectively. The atoms in a crystal are not static, but oscillate about their mean positions, usually by less than a few tenths of an angstrom. X-ray crystallography allows measuring the size of these oscillations.

Procedure

The technique of single-crystal X-ray
X-ray
crystallography has three basic steps. The first—and often most difficult—step is to obtain an adequate crystal of the material under study. The crystal should be sufficiently large (typically larger than 0.1 mm in all dimensions), pure in composition and regular in structure, with no significant internal imperfections such as cracks or twinning .

In the second step, the crystal is placed in an intense beam of X-rays, usually of a single wavelength (monochromatic X-rays), producing the regular pattern of reflections. As the crystal is gradually rotated, previous reflections disappear and new ones appear; the intensity of every spot is recorded at every orientation of the crystal. Multiple data sets may have to be collected, with each set covering slightly more than half a full rotation of the crystal and typically containing tens of thousands of reflections.

In the third step, these data are combined computationally with complementary chemical information to produce and refine a model of the arrangement of atoms within the crystal. The final, refined model of the atomic arrangement—now called a crystal structure —is usually stored in a public database.

Limitations

See also: Resolution (electron density)

As the crystal's repeating unit, its unit cell, becomes larger and more complex, the atomic-level picture provided by X-ray crystallography becomes less well-resolved (more "fuzzy") for a given number of observed reflections. Two limiting cases of X-ray crystallography—"small-molecule" (which includes continuous inorganic solids) and "macromolecular" crystallography—are often discerned. Small-molecule crystallography typically involves crystals with fewer than 100 atoms in their asymmetric unit ; such crystal structures are usually so well resolved that the atoms can be discerned as isolated "blobs" of electron density. By contrast, macromolecular crystallography often involves tens of thousands of atoms in the unit cell. Such crystal structures are generally less well-resolved (more "smeared out"); the atoms and chemical bonds appear as tubes of electron density, rather than as isolated atoms. In general, small molecules are also easier to crystallize than macromolecules; however, X-ray
X-ray
crystallography has proven possible even for viruses with hundreds of thousands of atoms. Though normally x-ray crystallography can only be performed if the sample is in crystal form, new research has been done into sampling non-crystalline forms of samples.

CRYSTALLIZATION

Further information: Crystallization
Crystallization
, Recrystallization (chemistry) § Single perfect crystals (for X-ray
X-ray
analysis) , and Protein crystallization A protein crystal seen under a microscope . Crystals used in X-ray
X-ray
crystallography may be smaller than a millimeter across.

Although crystallography can be used to characterize the disorder in an impure or irregular crystal, crystallography generally requires a pure crystal of high regularity to solve the structure of a complicated arrangement of atoms. Pure, regular crystals can sometimes be obtained from natural or synthetic materials, such as samples of metals , minerals or other macroscopic materials. The regularity of such crystals can sometimes be improved with macromolecular crystal annealing and other methods. However, in many cases, obtaining a diffraction-quality crystal is the chief barrier to solving its atomic-resolution structure.

Small-molecule and macromolecular crystallography differ in the range of possible techniques used to produce diffraction-quality crystals. Small molecules generally have few degrees of conformational freedom, and may be crystallized by a wide range of methods, such as chemical vapor deposition and recrystallization . By contrast, macromolecules generally have many degrees of freedom and their crystallization must be carried out so as to maintain a stable structure. For example, proteins and larger RNA
RNA
molecules cannot be crystallized if their tertiary structure has been unfolded ; therefore, the range of crystallization conditions is restricted to solution conditions in which such molecules remain folded. Three methods of preparing crystals, A: Hanging drop. B: Sitting drop. C: Microdialysis

Protein
Protein
crystals are almost always grown in solution. The most common approach is to lower the solubility of its component molecules very gradually; if this is done too quickly, the molecules will precipitate from solution, forming a useless dust or amorphous gel on the bottom of the container. Crystal
Crystal
growth in solution is characterized by two steps: nucleation of a microscopic crystallite (possibly having only 100 molecules), followed by growth of that crystallite, ideally to a diffraction-quality crystal. The solution conditions that favor the first step (nucleation) are not always the same conditions that favor the second step (subsequent growth). The crystallographer's goal is to identify solution conditions that favor the development of a single, large crystal, since larger crystals offer improved resolution of the molecule. Consequently, the solution conditions should disfavor the first step (nucleation) but favor the second (growth), so that only one large crystal forms per droplet. If nucleation is favored too much, a shower of small crystallites will form in the droplet, rather than one large crystal; if favored too little, no crystal will form whatsoever. Other approaches involves, crystallizing proteins under oil, where aqueous protein solutions are dispensed under liquid oil, and water evaporates through the layer of oil. Different oils have different evaporation permeabilities, therefore yielding changes in concentration rates from different percipient/protein mixture. The technique relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of percipient to prevent the diffusion of water out of the drop.

It is extremely difficult to predict good conditions for nucleation or growth of well-ordered crystals. In practice, favorable conditions are identified by screening; a very large batch of the molecules is prepared, and a wide variety of crystallization solutions are tested. Hundreds, even thousands, of solution conditions are generally tried before finding the successful one. The various conditions can use one or more physical mechanisms to lower the solubility of the molecule; for example, some may change the pH, some contain salts of the Hofmeister series or chemicals that lower the dielectric constant of the solution, and still others contain large polymers such as polyethylene glycol that drive the molecule out of solution by entropic effects. It is also common to try several temperatures for encouraging crystallization, or to gradually lower the temperature so that the solution becomes supersaturated. These methods require large amounts of the target molecule, as they use high concentration of the molecule(s) to be crystallized. Due to the difficulty in obtaining such large quantities (milligrams ) of crystallization-grade protein, robots have been developed that are capable of accurately dispensing crystallization trial drops that are in the order of 100 nanoliters in volume. This means that 10-fold less protein is used per experiment when compared to crystallization trials set up by hand (in the order of 1 microliter ).

Several factors are known to inhibit or mar crystallization. The growing crystals are generally held at a constant temperature and protected from shocks or vibrations that might disturb their crystallization. Impurities in the molecules or in the crystallization solutions are often inimical to crystallization. Conformational flexibility in the molecule also tends to make crystallization less likely, due to entropy. Ironically, molecules that tend to self-assemble into regular helices are often unwilling to assemble into crystals. Crystals can be marred by twinning , which can occur when a unit cell can pack equally favorably in multiple orientations; although recent advances in computational methods may allow solving the structure of some twinned crystals. Having failed to crystallize a target molecule, a crystallographer may try again with a slightly modified version of the molecule; even small changes in molecular properties can lead to large differences in crystallization behavior.

DATA COLLECTION

Mounting The Crystal

Play media Animation showing the five motions possible with a four-circle kappa goniometer. The rotations about each of the four angles φ, κ, ω and 2θ leave the crystal within the X-ray
X-ray
beam, but change the crystal orientation. The detector (red box) can be slid closer or further away from the crystal, allowing higher resolution data to be taken (if closer) or better discernment of the Bragg peaks (if further away).

The crystal is mounted for measurements so that it may be held in the X-ray
X-ray
beam and rotated. There are several methods of mounting. In the past, crystals were loaded into glass capillaries with the crystallization solution (the mother liquor ). Nowadays, crystals of small molecules are typically attached with oil or glue to a glass fiber or a loop, which is made of nylon or plastic and attached to a solid rod. Protein
Protein
crystals are scooped up by a loop, then flash-frozen with liquid nitrogen . This freezing reduces the radiation damage of the X-rays, as well as the noise in the Bragg peaks due to thermal motion (the Debye-Waller effect). However, untreated protein crystals often crack if flash-frozen; therefore, they are generally pre-soaked in a cryoprotectant solution before freezing. Unfortunately, this pre-soak may itself cause the crystal to crack, ruining it for crystallography. Generally, successful cryo-conditions are identified by trial and error.

The capillary or loop is mounted on a goniometer , which allows it to be positioned accurately within the X-ray
X-ray
beam and rotated. Since both the crystal and the beam are often very small, the crystal must be centered within the beam to within ~25 micrometers accuracy, which is aided by a camera focused on the crystal. The most common type of goniometer is the "kappa goniometer", which offers three angles of rotation: the ω angle, which rotates about an axis perpendicular to the beam; the κ angle, about an axis at ~50° to the ω axis; and, finally, the φ angle about the loop/capillary axis. When the κ angle is zero, the ω and φ axes are aligned. The κ rotation allows for convenient mounting of the crystal, since the arm in which the crystal is mounted may be swung out towards the crystallographer. The oscillations carried out during data collection (mentioned below) involve the ω axis only. An older type of goniometer is the four-circle goniometer, and its relatives such as the six-circle goniometer.

X-ray
X-ray
Sources

Further information: Diffractometer , Synchrotron light source , and Free-electron laser

Rotating Anode

Small scale can be done on a local X-ray
X-ray
tube source, typically coupled with an image plate detector. These have the advantage of being (relatively) inexpensive and easy to maintain, and allow for quick screening and collection of samples. However, the wavelength light produced is limited by anode material, typically copper . Further, intensity is limited by the power applied and cooling capacity available to avoid melting the anode. In such systems, electrons are boiled off of a cathode and accelerated through a strong electric potential of ~50 kV ; having reached a high speed, the electrons collide with a metal plate, emitting bremsstrahlung and some strong spectral lines corresponding to the excitation of inner-shell electrons of the metal. The most common metal used is copper , which can be kept cool easily, due to its high thermal conductivity , and which produces strong Kα and Kβ lines. The Kβ line is sometimes suppressed with a thin (~10 µm) nickel foil. The simplest and cheapest variety of sealed X-ray
X-ray
tube has a stationary anode (the Crookes tube
Crookes tube
) and run with ~2 kW of electron beam power. The more expensive variety has a rotating-anode type source that run with ~14 kW of e-beam power.

X-rays
X-rays
are generally filtered (by use of X-Ray Filters ) to a single wavelength (made monochromatic) and collimated to a single direction before they are allowed to strike the crystal. The filtering not only simplifies the data analysis, but also removes radiation that degrades the crystal without contributing useful information. Collimation is done either with a collimator (basically, a long tube) or with a clever arrangement of gently curved mirrors. Mirror systems are preferred for small crystals (under 0.3 mm) or with large unit cells (over 150 Å)

Synchrotron Radiation

Synchrotron radiation are some of the brightest lights on earth. It is the single most powerful tool available to X-ray
X-ray
crystallographers. It is made of X-ray
X-ray
beams generated in large machines called synchrotrons . These machines accelerate electrically charged particles, often electrons, to nearly the speed of light and confine them in a (roughly) circular loop using magnetic fields.

Synchrotrons are generally national facilities, each with several dedicated beamlines where data is collected without interruption. Synchrotrons were originally designed for use by high-energy physicists studying subatomic particles and cosmic phenomena. The largest component of each synchrotron is its electron storage ring. This ring is actually not a perfect circle, but a many-sided polygon. At each corner of the polygon, or sector, precisely aligned magnets bend the electron stream. As the electrons’ path is bent, they emit bursts of energy in the form of X-rays.

Using synchrotron radiation frequently has specific requirements for X-ray
X-ray
crystallography. The intense ionizing radiation can cause radiation damage to samples, particularly macromolecular crystals. Cryo crystallography protects the sample from radiation damage, by freezing the crystal at liquid nitrogen temperatures (~100 K ). However, synchrotron radiation frequently has the advantage of user selectable wavelengths, allowing for anomalous scattering experiments which maximizes anomalous signal. This is critical in experiments such as SAD and MAD .

Free Electron
Electron
Laser

Recently, free electron lasers have been developed for use in X-ray crystallography. These are the brightest X-ray
X-ray
sources currently available; with the X-rays
X-rays
coming in femtosecond bursts. The intensity of the source is such that atomic resolution diffraction patterns can be resolved for crystals otherwise too small for collection. However, the intense light source also destroys the sample, requiring multiple crystals to be shot. As each crystal is randomly oriented in the beam, hundreds of thousands of individual diffraction images must be collected in order to get a complete data-set. This method, serial femtosecond crystallography, has been used in solving the structure of a number of protein crystal structures, sometimes noting differences with equivalent structures collected from synchrotron sources.

Recording The Reflections

An X-ray diffraction
X-ray diffraction
pattern of a crystallized enzyme. The pattern of spots (reflections) and the relative strength of each spot (intensities) can be used to determine the structure of the enzyme.

When a crystal is mounted and exposed to an intense beam of X-rays, it scatters the X-rays
X-rays
into a pattern of spots or reflections that can be observed on a screen behind the crystal. A similar pattern may be seen by shining a laser pointer at a compact disc . The relative intensities of these spots provide the information to determine the arrangement of molecules within the crystal in atomic detail. The intensities of these reflections may be recorded with photographic film , an area detector or with a charge-coupled device (CCD) image sensor. The peaks at small angles correspond to low-resolution data, whereas those at high angles represent high-resolution data; thus, an upper limit on the eventual resolution of the structure can be determined from the first few images. Some measures of diffraction quality can be determined at this point, such as the mosaicity of the crystal and its overall disorder, as observed in the peak widths. Some pathologies of the crystal that would render it unfit for solving the structure can also be diagnosed quickly at this point.

One image of spots is insufficient to reconstruct the whole crystal; it represents only a small slice of the full Fourier transform. To collect all the necessary information, the crystal must be rotated step-by-step through 180°, with an image recorded at every step; actually, slightly more than 180° is required to cover reciprocal space, due to the curvature of the Ewald sphere . However, if the crystal has a higher symmetry, a smaller angular range such as 90° or 45° may be recorded. The rotation axis should be changed at least once, to avoid developing a "blind spot" in reciprocal space close to the rotation axis. It is customary to rock the crystal slightly (by 0.5–2°) to catch a broader region of reciprocal space.

Multiple data sets may be necessary for certain phasing methods. For example, MAD phasing requires that the scattering be recorded at least three (and usually four, for redundancy) wavelengths of the incoming X-ray
X-ray
radiation. A single crystal may degrade too much during the collection of one data set, owing to radiation damage; in such cases, data sets on multiple crystals must be taken.

DATA ANALYSIS

Crystal
Crystal
Symmetry, Unit Cell, And Image Scaling

Further information: Space group
Space group

The recorded series of two-dimensional diffraction patterns, each corresponding to a different crystal orientation, is converted into a three-dimensional model of the electron density; the conversion uses the mathematical technique of Fourier transforms, which is explained below . Each spot corresponds to a different type of variation in the electron density; the crystallographer must determine which variation corresponds to which spot (indexing), the relative strengths of the spots in different images (merging and scaling) and how the variations should be combined to yield the total electron density (phasing).

Data processing begins with indexing the reflections. This means identifying the dimensions of the unit cell and which image peak corresponds to which position in reciprocal space. A byproduct of indexing is to determine the symmetry of the crystal, i.e., its space group . Some space groups can be eliminated from the beginning. For example, reflection symmetries cannot be observed in chiral molecules; thus, only 65 space groups of 230 possible are allowed for protein molecules which are almost always chiral. Indexing is generally accomplished using an autoindexing routine. Having assigned symmetry, the data is then integrated. This converts the hundreds of images containing the thousands of reflections into a single file, consisting of (at the very least) records of the Miller index of each reflection, and an intensity for each reflection (at this state the file often also includes error estimates and measures of partiality (what part of a given reflection was recorded on that image)).

A full data set may consist of hundreds of separate images taken at different orientations of the crystal. The first step is to merge and scale these various images, that is, to identify which peaks appear in two or more images (merging) and to scale the relative images so that they have a consistent intensity scale. Optimizing the intensity scale is critical because the relative intensity of the peaks is the key information from which the structure is determined. The repetitive technique of crystallographic data collection and the often high symmetry of crystalline materials cause the diffractometer to record many symmetry-equivalent reflections multiple times. This allows calculating the symmetry-related R-factor , a reliability index based upon how similar are the measured intensities of symmetry-equivalent reflections, thus assessing the quality of the data.

Initial Phasing

Further information: Phase problem

The data collected from a diffraction experiment is a reciprocal space representation of the crystal lattice. The position of each diffraction 'spot' is governed by the size and shape of the unit cell, and the inherent symmetry within the crystal. The intensity of each diffraction 'spot' is recorded, and this intensity is proportional to the square of the structure factor amplitude . The structure factor is a complex number containing information relating to both the amplitude and phase of a wave . In order to obtain an interpretable electron density map, both amplitude and phase must be known (an electron density map allows a crystallographer to build a starting model of the molecule). The phase cannot be directly recorded during a diffraction experiment: this is known as the phase problem . Initial phase estimates can be obtained in a variety of ways:

* AB INITIO PHASING or DIRECT METHODS – This is usually the method of choice for small molecules ( F c a l l r e f l e c t i o n s F o {displaystyle R={frac {sum _{mathrm {all reflections} }leftF_{o}-F_{c}right}{sum _{mathrm {all reflections} }leftF_{o}right}}}

where F is the structure factor . A similar quality criterion is Rfree, which is calculated from a subset (~10%) of reflections that were not included in the structure refinement. Both R factors depend on the resolution of the data. As a rule of thumb, Rfree should be approximately the resolution in angstroms divided by 10; thus, a data-set with 2 Å resolution should yield a final Rfree ~ 0.2. Chemical bonding features such as stereochemistry, hydrogen bonding and distribution of bond lengths and angles are complementary measures of the model quality. Phase bias is a serious problem in such iterative model building. Omit maps are a common technique used to check for this.

It may not be possible to observe every atom in the asymmetric unit. In many cases, disorder smears the electron density map. Weakly scattering atoms such as hydrogen are routinely invisible. It is also possible for a single atom to appear multiple times in an electron density map, e.g., if a protein sidechain has multiple (

Links: ------ /wiki/Crystal /wiki/Atom /wiki/X-rays /wiki/Diffraction

.