6 Introduction:1X-ray scattering techniques are a family of non-destructive analytical techniques which reveal information about the crystallographic structure, chemical composition, and physical properties of materials and thin films.These techniques are based on observing the scattered intensity of an x-ray beam hitting a sample as a function of incident and scattered angle, polarization, and wavelength or energy.To characterize the crystallographic structure of crystalline material and powdered solid samples different diffraction techniques are used
7 Introduction:2Single-crystal X-ray diffraction is a technique used to solve the complete structure of crystalline materials, ranging from simple inorganic solids to complex macromolecules, such as proteins.Powder diffraction (XRD) is a technique use to characterize the crystallographic structure, crystallite size (grain size), and preferred orientation in polycrystalline or powdered solid samples.
8 X-ray Scattering Techniques:1 X-ray scattering techniques are a family of non-destructive analytical techniques.X-ray scattering techniques which reveal information about the crystallographic structure, chemical composition, and physical properties of materials and thin films.These techniques are based on observing the scattered intensity of an x-ray beam.The X-ray beam hits the sample as a function of incident and scattered angle, polarization, and wavelength or energy.
9 X-ray Scattering Techniques:2 X-ray diffraction techniques are based on the elastic scattering of x-rays from structures that have long range order.The most comprehensive description of scattering from crystals is given by the dynamical theory of diffraction.Max von Laue, in 1912, discovered that crystalline substances act as three-dimensional diffraction gratings for X-ray wavelengths similar to the spacing of planes in a crystal lattice.
10 X-ray Diffraction Pattern This is an X-ray diffraction pattern formed when X-rays are focused on a crystalline material.Each dot, called a reflection, forms from the coherent interference of scattered X-rays passing through the crystal.
11 Single-crystal X-ray Diffraction-1 Single-crystal X-ray Diffraction is a non-destructive analytical technique.Single crystal X-ray diffraction provides detailed information about the internal lattice of crystalline substances.Single crystal X-ray diffraction provide details about unit cell dimensions, bond-lengths, bond-angles, and details of site-ordering.Single crystal X-ray diffraction is directly related is single-crystal refinement.In single-crystal refinement, the data generated from the X-ray analysis is interpreted and refined to obtain the crystal structure.
12 Single Crystal X-ray Diffraction-2 The most common experimental method of obtaining a detailed structure of a molecule is single crystal X-ray diffraction (SXRD) .Single crystal X-ray diffraction (SXRD) allows resolution of individual atoms.single crystal X-ray diffraction (SXRD) performed by analyzing the pattern of X-rays diffracted by an ordered array of many identical molecules (single crystal).Many pure compounds, from small molecules to organometallic complexes, proteins, and polymers, solidify into crystals under the proper conditions.
13 Single Crystal X-ray Diffraction-3 When solidifying into the crystalline state, these individual molecules typically adapt one of the few possible 3D orientations.When a monochromatic X-ray beam is passed through a single crystal, the radiation interacts with the electrons in the atoms, resulting in scattering of the radiation to produce a unique image pattern.Multiple images are recorded, with an area X-ray detector, as the crystal is rotated in the X-ray beam.Computationally intensive analysis of a set images results in a solution for the 3D structure of the molecule.
14 Principles of X-ray Diffraction:1 X-ray diffraction is based on constructive interference of monochromatic X-rays and a crystalline sample.These X-rays are generated by a cathode ray tube, filtered to produce monochromatic radiation, collimated to concentrate, and directed toward the sample.The interaction of the incident rays with the sample produces constructive interference (and a diffracted ray) when conditions satisfy Bragg's Law (nλ=2d sinθ).
15 Principles of X-ray Diffraction:2 This law relates the wavelength of electromagnetic radiation to the diffraction angle and the lattice spacing in a crystalline sample.These diffracted X-rays are then detected, processed and counted.By changing the geometry of the incident rays, the orientation of the centered crystal and the detector, all possible diffraction directions of the lattice should be attained.
16 Principles of X-ray Diffraction:3 All diffraction methods are based on generation of X-rays in an X-ray tube. These X-rays are directed at the sample, and the diffracted rays are collected.A key component of all diffraction is the angle between the incident and diffracted rays.Powder and single-crystal diffraction vary in instrumentation.
17 Instrumentation of X-ray Diffraction X-ray diffractometers consist of three basic elements, an X-ray tube, a sample holder, and an X-ray detector.X-rays are generated in a cathode ray tube by heating a filament to produce electrons, accelerating the electrons toward a target by applying a voltage, and impact of the electrons with the target material.When electrons have sufficient energy to dislodge inner shell electrons of the target material, characteristic X-ray spectra are produced.X-rays must be produced using a synchotron, which emits a much stronger beam.
18 How Does It Work?These spectra consist of several components, the most common being Kα and Kβ.Kα consists, in part, of Kα1 and Kα2.Kα1 has a slightly shorter wavelength and twice the intensity as Kα2.In case of Molybdenum, which is a common target material for single-crystal diffraction,Kα radiation = Å.
19 How Does It Work - 1For diffraction, monochromatic X-rays are needed which are produced by filtering, by foils or crystal.Kα1and Kα2 are sufficiently close in wavelength such that a weighted average of the two is used.The specific wavelengths are characteristic of the target material.
20 How Does It Work - 3When the geometry of the incident X-rays impinging the sample satisfies the Bragg Equation, constructive interference occurs.A detector records and processes this X-ray signal and converts the signal to a count rate which is then output to a device such as a printer or computer monitor.Modern single-crystal diffractometers use CCD (charge-coupled device) technology to transform the X-ray photons into an electrical signal which are then sent to a computer for processing.
21 How Does It Work - 4Single-crystal diffractometers use either 3- or 4-circle goniometers.These circles refer to the four angles (2θ, χ, φ, and Ω) that define the relationship between the crystal lattice, the incident ray and detector.Samples are mounted on thin glass fibers which are attached to brass pins and mounted onto goniometer heads.Adjustment of the X, Y and Z orthogonal directions allows centering of the crystal within the X-ray beam.
22 How Does It Work - 5X-rays leave the collimator and are directed at the crystal.Rays are either transmitted through the crystal, reflected off the surface, or diffracted by the crystal lattice.A beam stop is located directly opposite the collimator to block transmitted rays and prevent burn-out of the detector.Reflected rays are not picked up by the detector due to the angles involved.Diffracted rays at the correct orientation for the configuration are then collected by the detector.
23 Applications - 1Single-crystal X-ray diffraction is most commonly used for precise determination of a unit cell, including cell dimensions and positions of atoms within the lattice.Bond-lengths and angles are directly related to the atomic positions.The crystal structure of a mineral is a characteristic property that is the basis for understanding many of the properties of each mineral.
24 Applications - 2New mineral identification, crystal solution and refinement.Determination of unit cell, bond-lengths, bond-angles and site-ordering.Characterization of cation-anion coordination.Variations in crystal lattice with chemistry.
25 Strengths X-ray Diffraction No separate standards requiredNon-destructiveDetailed crystal structure, including unit cell dimensions, bond-lengths, bond-angles and site-ordering informationDetermination of crystal-chemical controls on mineral chemistryWith specialized chambers, structures of high pressure and/or temperature phases can be determined.Powder patterns can also be derived from single-crystals by use of specialized cameras (Gandolfi)
26 Limitations X-ray Diffraction Must have a single, robust (stable) sample, generally between 50–250 microns in size.Optically clear sample .Twinned samples can be handled with difficulty .Data collection generally requires between 24 and 72 hours
27 Molecular Replacement (MR)-1 In cases where the crystal under investigation is isomorphous with the known one (having the same space group and cell constants within experimental error), analysis can proceed directly by difference Fourier methods.However, more commonly, isomorphism does not exist, and it becomes necessary to seek other ways of utilizing the known structure information to facilitate the target structure determination.To this end, the Molecular Replacement (MR) method has proved to be particularly successful
28 Molecular Replacement (MR)-2 The prerequisites for using the MR method are:an observed diffraction pattern - intensities - for the unknown structure, or the target;the atomic coordinates of an homologous protein structure, or the probe.The MR task involves positioning the probe within the unit cell of the target crystal in such a way that the theoretical diffraction pattern that would result from this model closely matches the experimental one.
29 Molecular Replacement (MR)-3 One molecule is present in the asymmetric unit, six parameters (three rotational and three translational), describe how the probe is placed in the unit cell.Study on these six parameters determine the position of the probe that gives the best agreement between observed and calculated structure factor.This study needs too much computation.
30 Molecular Replacement Method-1 The first is the determination of the correct orientation of the probe, and the second is the determination of the position of the correctly-oriented molecule within the unit cell.From the theoretical analysis of the properties of the Patterson function it became obvious that such a six-parameter search could be reduced to two three-dimensional problems.
31 Molecular Replacement Method-2 We have a probe molecule A and the unknown molecule A' similar to A.The position of A' is different from A.To superimpose the molecule A with A' we have, firstly, to apply the rotation R, and then the translation T.Therefore, the main aim of the MR method is to find these two operators, or, in other terms, to solve the Rotation and the Translation functions.This figure shows a pictorial representationof the MR problem.
32 Molecular Replacement Method-3 Factors affectingSolving a structure by the molecular replacement method is not always a straight forward task.Depending on the complexity of the problem, a number of important factors must be taken into consideration in order to ensure a correct structure determination.The factors affecting the molecular replacement solution are crystal and X-ray data.
33 Factors affecting MR - 1 The crystal The crystal form most favorable for the molecular replacement method is the one with only a single molecule in the asymmetric unit.The presence of multiple copies of the protein in the asymmetric unit reduces the signal-to-noise ratio for the correct peaks on the rotation and translation function.Furthermore, the presence of non-crystallographic symmetry between these copies can introduce difficulties in determining correct solutions.However, there are many cases in which the structure was solved by molecular replacement method where there are several molecules in the asymmetric unit.
34 Factors affecting MR - 2 The X-ray data It is very important that the experimental data are as complete as possible, ideally 100%.The data should be of high quality.Possible problems include:systematically missing regions,ice ring,detector overloads, etc.
35 Molecular Replacement Search Probe The choice of a search probe has to be made by considering possible structural similarities with the target molecule (within Å r.m.s.d.).The two structures should have, at least, around % amino-acid sequence identity and, in general, the higher the better.Also, when the choice for the search probe is given between a crystallographic structure and an NMR structure, the former, normally, provides a higher degree of success.
36 Molecular Replacement Search Probe This has mainly to do the imprecision of the NMR protein model.Furthermore, the search model does not necessarily have to be used in its integrity; some parts of it can be removed completelyor the side chains of some residues can be trimmed to alanine/glycine residues in the regions where the largest differences with the unknown structure are expected.
37 What You Learn… You have learnt : X-ray scattering techniques. Principles of Single-crystal X-ray DiffractionInstrumentation used for Single-crystal X-ray DiffractionApplications of Single-crystal X-ray Diffraction Oparins HypothesisStrengths and Limitations of Single-crystal X-ray Diffraction
40 Study Tips - 1 Book Book Book Title:Introduction to Mineral Sciences. Author: Putnis, ACambridge University PressBookTitle: Biological ScienceAuthor: Taylor, Green & StoutBookTitle: ABC Of BiologyPublisher :Holy Faith
41 Study Tips - 2 www.en.wikipedia.org Microsoft Encarta Encyclopedia Introduction to Xray Diffraction from Links for Mineralogists, Institute of Mineralogy, University of Wuerzburg.An Introduction to the Scope, Potential and Applications of X-ray Analysis, from the International Union of Crystallography