Name: reading crystal patterns with x-ray diffraction
Uploaded: 2023-04-30T11:59:10.000Z
Duration: 1 min 10 s
Channel: JoVE

X-ray diffraction or XRD is a non-destructive analytical technique used to study the structural and spatial arrangement of atoms. It is commonly used to study biological samples such as protein crystals, nucleic acids, and ribosomes. It is also used to study crystalline powders of pharmaceutical formulations.
The XRD analyzer consists of a source of highly focused X-ray beams that strike the sample, like a crystalline solid.
These X-rays diffract, producing an interference pattern that is recorded by an electronic detector.
Early insights into the DNA double-helical structure were obtained using XRD, which shows the X-shaped diffraction pattern and other dimensional details of the DNA helix.
Single-crystal XRD is used in protein crystallography to determine its three-dimensional structure.
In powder XRD, a polycrystalline sample is seen as concentric rings. The phase and purity of the material can be determined by comparison with the database.

x-ray diffraction of biological samples

X-ray Diffraction of Biological Samples

X-ray diffraction or XRD is an analytical tool that utilizes X-rays to study ordered structures such as crystalline organic and inorganic samples, polycrystalline materials, proteins, carbohydrates, and drugs.
According to Bragg&#39;s law, when X-rays strike the sample positioned on a stage, the rays are&#160; scattered by the electron clouds around the sample atoms. The&#160; X-ray diffraction or scattering is caused by constructive interference of the X-ray waves that reflect off the internal crystal planes. The scattering angle of the rays in specific directions by the atoms within the sample provides a diffraction pattern representing the electron density due to the atoms and bonds within the crystal. This information about the crystal lattice arrangement helps identify the material from its diffraction pattern, which resembles&#160; a fingerprint. The material can be identified by comparing this fingerprint with a database, such as&#160; the International Center for Diffraction Database (ICDD). XRD can look at the size, shape, and internal structure and help in the basic structural determination of a sample or unknown material.
For a single crystal, a typical diffraction pattern comprises spots that are 2D slices of 3-dimensional spheres. A computer program can integrate the resulting spots to determine the shape and intensity of the diffracted X-rays. Whereas, in a powder sample, the X-rays interact with many tiny crystals in random orientations. Hence, instead of spots, a circular diffraction pattern is observed. The intensities of the diffracted circles are plotted against the angles between the ring of the beam axis, giving a 2D plot known as a powder pattern. Amorphous or non-crystalline powders give a broad peak due to scattering from different directions compared to ordered scattering in crystalline powders, giving sharp concentric rings.
Certain precautions should be taken while studying biological samples using this technique. First of all, well-ordered crystals are a prerequisite for deciphering the protein structure through single-crystal XRD. Generally, single small crystals with well-defined faces, if they contain heavier atoms, will suffice and give a good diffraction pattern. For organic compounds, however, the crystals need to be larger. Without well-defined, viable crystals, this technique is not feasible. Some molecules are inherently more crystalline than others; thus, the difficulty of obtaining X-ray quality crystals can vary between compounds and is the main limiting factor of this technique. Further, the powder samples should be fine powder and have no clumps.

X-ray diffraction or XRD is an analytical tool that utilizes X-rays to study ordered structures such as crystalline organic and inorganic samples, ...

Education

JoVE Core

Cell Biology

Unit 1

Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

reading crystal patterns with x-ray diffraction

X-ray diffraction, or XRD, is used to study ordered structures in crystalline organic and inorganic samples, polycrystalline materials, proteins, carbohydrates, and drugs. The method shows how atoms are arranged in a sample by measuring how X-rays scatter from it.

When X-rays hit a sample on a stage, the beams interact with the electron clouds around the atoms. The scattering follows Bragg’s law, and it comes from constructive interference of X-ray waves reflected from internal crystal planes. The result is a diffraction pattern that reflects the electron density from the atoms and bonds in the crystal.

This pattern acts like a fingerprint for the material. Scientists can compare it with a database such as the International Center for Diffraction Database, or ICDD, to help identify an unknown sample. XRD also helps determine size, shape, and internal structure in basic structural analysis.

Single crystals and powder samples give different patterns. A single crystal produces spots that are 2D slices of 3-dimensional spheres, and computer software can integrate those spots to find the shape and intensity of the diffracted X-rays. Powder samples contain many tiny crystals in random directions, so they produce circular rings instead of spots. The ring intensities are plotted against the angle from the beam axis to make a 2D powder pattern. Amorphous, or non-crystalline, powders give broad peaks, while crystalline powders give sharp concentric rings.

Biological samples need careful preparation for XRD. Single-crystal XRD depends on well-ordered crystals, and single small crystals with clear faces can work well, especially if they contain heavier atoms. Organic compounds usually need larger crystals. Without viable crystals, the method is not feasible, and the difficulty of growing X-ray quality crystals often becomes the main limit. Powder samples should also be very fine and free of clumps.

Reading Crystal Patterns with X-Ray Diffraction

reading-crystal-patterns-with-x-ray-diffraction

overview of cell separation and isolation

overview-of-cell-separation-and-isolation

Methods for Separating Specific Cell Types

Cell separation is used to isolate one cell type from a mixed sample. Early work in this field began in 1964, when S. H. Seal separated large tumor cells from smaller blood cells by filtration. Two years later, Pohl and Hawk showed that different cell types can respond differently to a nonuniform electric field. These findings helped launch modern cell separation methods. Several common techniques are used to separate cells. Filtration removes cells based on size, while density gradient...

cell culture

Growing Cells in the Lab

Growing cells in the lab depends on the right culture type and the right growth conditions. Most vertebrate cells grow attached to a surface as adherent cultures. These cells are grown in flasks and plates that are chemically treated to help the cells attach. Some cells, such as hematopoietic cells, grow in suspension instead. Suspension cultures can be kept in non-treated cultureware with magnetic stirrers or spinner flasks to keep the medium moving. The growth medium is a key part of...

cell lines

How Cell Lines Change and Are Verified

Cell lines are populations of cells grown in vitro that can be subcultured over many generations. Normal cells eventually stop dividing after a set number of cell divisions. This stopping point is called replicative senescence. The division limit is known as the Hayflick limit. Leonard Hayflick described this limit in 1961 after he found that fetal cells in culture could divide only about 40 to 60 times. Telomeres help explain why this happens. Telomeres shorten with each round of cell...

hybridoma technology

Monoclonal Antibody Production with Hybridomas

Hybridoma technology makes monoclonal antibodies in large amounts. Monoclonal antibodies are antibodies that bind to just one antigenic determinant, or epitope. These antibodies are important in research, diagnostics, and disease therapy. The method was developed in 1975 by Georges Köhler and Cesar Milstein, and it received the Nobel Prize in Medicine in 1984 for changing research and treatment. The process starts by fusing cells with different strengths. Common fusion methods include...

tissue homogenization and cell lysis

Lysis Methods for Preparing Tissue Samples

Tissue sample preparation often starts with homogenization and cell lysis. This step breaks tissue structure and opens cells so their parts can be studied. The best method depends on the sample type, the amount of sample, the analyte to be collected, and how sensitive the method is. Tissue homogenization methods are usually grouped into mechanical and non-mechanical methods. Mechanical methods use external physical force to disrupt tissues and cells. These methods may use grinding, shearing,...

subcellular fractionation

Separating Cell Organelles by Centrifugation

Separating cell organelles by centrifugation lets scientists isolate membrane-bound parts of a cell after lysis. The starting material is a homogenate, which is the mixture left after cells are broken open. These isolated fractions help researchers study specific cell parts, measure protein activity in one location, and support some diagnostic tests. Fractionation usually uses centrifugation, which spins the sample to separate its contents. The two main methods are differential centrifugation...

flow cytometry

Flow Cytometry in Cell Sorting

Flow cytometry in cell sorting tracks cells as they move through a laser beam. The instrument measures light scatter and fluorescence, then uses those signals to separate cells based on their properties. It is also used to count cells and study cell populations in detail. The method began in 1934 with Andrew Moldavan, a bacteriologist who counted cells in a flowing capillary system. He pushed cells through a capillary tube and viewed them under a microscope. Later, photometry made it possible...

principles of column chromatography

Column Chromatography: Separation Factors

Column chromatography separates compounds in a sample mixture by passing a solvent through a packed column. The technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, who used organic solvents to separate plant pigments. In 1941, Archer John Porter Martin and R. L. M. Synge improved the method by packing silica gel into a column and separating amino acids with a chloroform-water mobile phase. That work became the first report on column chromatography. Today, column...

types of column chromatography

Column Chromatography for Protein Purification

Column chromatography is used to purify proteins by matching the column material to the sample. The stability and compatibility of the material matter because pH, temperature, and solvent can change how the packed column performs. The right column choice helps improve purification efficiency and can be adjusted for the protein being isolated. Gel filtration chromatography separates proteins by size when the protein’s chemical nature is unknown. It uses matrices such as agarose, polyacrylamide,...

immunoprecipitation

Protein Capture with Antibodies

Immunoprecipitation uses antibodies to capture a specific protein or protein complex from a sample. The protein stays in its native state, which helps scientists study protein-protein interactions, quaternary structure, and larger supramolecular complexes. Several related methods build on this idea. Chromatin immunoprecipitation, or ChIP, is used to study protein-DNA and protein-RNA interactions. Cross-linking immunoprecipitation, or CLIP, is used to find where proteins bind endogenous RNA...

tagging and fusion proteins

Protein Tags for Purification and Detection

Protein tags help scientists isolate and study a specific protein from the other proteins in a cell. The target gene is overexpressed in a suitable host so the cell makes large amounts of the protein of interest. A tag, or label, is added to the gene to create a fusion protein that combines the target protein with the tag. These tags make detection and purification easier. Common tag types include affinity tags, epitope tags, reporter tags, fluorescent tags, and self-splicing intein tags. In...

sds-page

Protein Size Separation by SDS-PAGE

SDS-PAGE separates proteins by size using a polyacrylamide gel and an electric field. It is a type of gel electrophoresis, which moves biological macromolecules through a gel matrix. In PAGE, proteins pass through polyacrylamide gel and can be separated by size, charge, or both, depending on the method used. In native PAGE, intact proteins keep their natural charges and are separated by both net charge and size. In SDS-PAGE, proteins are first denatured and coated with sodium dodecyl sulfate,...

western blotting

Western Blotting for Protein Detection

Western blotting is a protein detection method used in biology and medicine. It helps identify proteins in biological samples and can support disease detection, including bovine spongiform encephalopathy, human immunodeficiency virus, and feline immunodeficiency virus. The process starts with SDS-PAGE, which stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This step separates proteins in a sample before they are moved to a membrane. The proteins are then transferred to...

two-dimensional gel electrophoresis

Protein Separation by Charge and Mass

Two-dimensional gel electrophoresis separates proteins by charge first and then by mass. It was first introduced by O’Farrell and Klose in 1975. Because it uses two separating steps, it gives a higher-resolution result than one-dimensional gel electrophoresis. The first dimension uses isoelectric focusing, or IEF, on immobilized pH gradient, or IPG, strips. Before this step, biological samples such as cells and tissues are treated with a buffer that contains urea, dithiothreitol, detergents,...

enzyme-linked immunosorbent assay

ELISA Test Types and Signal Detection

ELISA is an antibody-based test used to detect and measure specific molecules in a sample. It was developed in 1971 by Peter Perlman and Eva Engvall. Unlike western blot, ELISA is done in microtiter plates or in vivo rather than on an absorbent membrane. In ELISA, an antibody has an enzyme attached to its constant region. The variable region stays free to bind a matching antigen. After binding, an enzyme-substrate reaction makes the target visible or measurable. In many tests, the substrate is...

maldi-tof mass spectrometry

MALDI-TOF for Rapid Sample Analysis

MALDI-TOF mass spectrometry analyzes compounds by their mass-to-charge ratio, or m/z. It is a powerful way to detect and separate many chemical and biological samples. A mass spectrometer has three main parts: an ion source, a mass analyzer, and a detector. These parts work together, and the design can change depending on the source and analyzer used. Matrix-assisted laser desorption ionization, or MALDI, is a common ion source in these instruments. It creates soft ionization, which helps...

peptide identification using tandem mass spectrometry

peptide-identification-using-tandem-mass-spectrometry

Reading Peptide Fragments with Tandem MS

Tandem mass spectrometry helps identify peptides by measuring their mass and breaking them into smaller pieces. It is also called MS/MS or MS2. The method uses two mass analyzers to isolate a biomolecule and study its chemical properties. This approach is important in proteomics, the study of proteins on a large scale. Peptides from a complex mixture are often first separated as much as possible after enzymatic digestion. Instruments such as gel electrophoresis or liquid chromatography can...

X-ray diffraction, or XRD, is used to study ordered structures in crystalline organic and inorganic samples, polycrystalline materials, proteins, carbohydrates, and drugs. The method shows how atoms are arranged in a sample by measuring how X-rays scatter from it. When X-rays hit a sample on a stage, the beams interact with the electron clouds around the atoms. The scattering follows Bragg’s law, and it comes from constructive interference of X-ray waves reflected from internal crystal planes.

applications of nmr in biology

NMR Spectroscopy in Biology and MRI

Nuclear magnetic resonance (NMR) spectroscopy is a useful analytical tool in biology. Researchers have used it for more than 50 years. F. Bloch and E. Purcell developed NMR in 1946 and received the 1952 Nobel Prize in Physics for this work. NMR studies biological macromolecules and small molecules. These include proteins, nucleic acids, lipids, and organic compounds such as pharmaceutical drugs. The technique works by using the magnetic properties of certain nuclei. The basic idea behind NMR...

proteomics

Proteomics and Protein Expression

Proteomics studies the proteins made by a cell type and how those proteins function. A proteome is the full set of proteins produced by a cell type, and proteomics is the large-scale study of that protein set. The term was coined by Mark Wilkins, who described it as the “PROTein complement expressed by a genOME.” Proteomics is closely linked to genomics because genes lead to mRNAs, and mRNAs usually encode proteins. However, not every mRNA is translated into a protein. This means mRNA data can...

key terms and concepts

Learn how X-ray diffraction reveals crystal structure in proteins, drugs, and other materials by analyzing spots, rings, and powder patterns.

Reading Crystal Patterns with X-Ray Diffraction | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Reading Crystal Patterns with X-Ray Diffraction

Description

Transcript

Tags