Andrew Chen Xraycrystallography

8/3/2019 Andrew Chen Xraycrystallography

1/9

X-Ray Crystallography a Concise Summary of its Uses and its Function

Andrew Chen (603231132)Physics 89

Abstract

X-Ray crystallography is the primary technique in which one can observe detailed three

dimensional structures of molecules or atoms, especially molecules or atoms that pertain

to living organisms and systems. X-Ray crystallography utilizes the small wavelengths

of X-rays to, in essence, see what the larger wavelengths of visible light cannot.

Because an atom/molecule is usually not very much bigger than 10-10 meters, for

example, the distance between to base pairs of DNA are two nanometers apart; visible

light has wavelengths far to large to see them (400 nanometers to 700 nanometers). It is

usually standard that in order to see an object, the size of that particular object has to be

at least half of the wavelength of the light being used to see it. This is why X-Ray

crystallography must be used to see molecules and atoms.

Introduction

In the past, it would have been impossible for people to observe molecules and atoms,

however, now X-ray crystallography and other tools such as an electron microscope can

achieve this previously unachievable feat. As stated above, with the use of X-rays, one

can observe these miniscule particles and see the structures that are within them. In the

following couple of pages, the process in which X-rays are used to provide an image of a

particle will be explained.

Crystallization

X-ray crystallography works by shooting X-rays at the molecule or atom and using the

resulting diffraction pattern of the X-rays to see what the molecular structure of the


2/9

molecule or atom is. In order for the X-rays to diffract uniformly or predictably, the atom

or molecule itself must have some type of spatial uniformity. Without this uniformity,

the atom or molecule itself will scatter the X-rays in an unpredictable manner making it

difficult to collect data. In order to achieve this uniformity, one must crystallize the atom

or molecule so that it will diffract the X-rays in a way that is understandable. Once the

atom or molecule is crystallized, one can shoot X-rays onto the atom or molecule and the

diffraction pattern will be captured on either film (what was first used) or by a CCD

detector (what is currently being used).

Image of Crystallized Atoms Image of X-Ray Diffraction

After the diffraction pattern is captured, the image is processed by a computer and

the structure of the atom or molecule can be attained. The X-ray diffraction pattern

appears to be a picture of dots in a circle, however the position of the dots in respect to

other dots give information to what the structure of the molecule actually is. There is a

complex formula that can be done by hand to figure out what each dot and/or


3/9

combination of dots corresponds to, however that formula is very complex and it is

standard to have a computer form the image. Below is an image depicting a X-ray

diffraction pattern of myoglobin, a protein found in muscles that bind with molecular

oxygen, and what the actual structure of myoglobin is. Today computers are used to

form these three dimensional images from data obtained by X-ray crystallography.

FFT represents the function needed to This is an X-ray diffraction pattern of This is the structure of myoglobin

convert the dot pattern to an image, myoglobin (note this is not the correspondingFFT-1 represents the inverse of that image to the X-ray diffraction

function. on the left, its here to serve the

purpose of showing the significantdifferences between the diffraction

pattern and the structure of the

molecule.
http://www.answers.com/main/ntquery?method=4&dsname=Wikipedia+Images&dekey=Myoglobindiffraction.png


4/9

This is what the a standard X-ray crystallography apparatus should look like. The images on the bottom are pictures of actual X-ray

diffraction apparatuses.

What X-ray Crystallography is Used for

One of the, if not the most famous discoveries made with X-ray crystallography was the

discovery of the structure of DNA. Two scientists, Maurice Wilkins (1916-2004) and

Rosalind Franklin (1920-1958), worked together and used X-ray crystallography to form

a diffraction pattern of DNA. However, it was James Watson (1928- ) and Francis Crick

(1916-2004), who were actively working on the structure of DNA together at the time

(and they also worked with X-ray crystallography and understood to a good extent how it


5/9

functioned), that took the excellent X-ray images taken by Franklin, and deduced the

final and correct structure of DNA. Together the four scientists announced publicly the

structure of DNA in an issue of the popular biological periodicalNature. In 1962,

Watson, Crick, and Wilkins got the Nobel Prize in medicine for their discovery of the

structure of DNA. Unfortunately, Franklin was not alive at the time to receive the award,

she died at before the honor was given out. Without X-ray crystallography, Watson and

Crick would not have been able to confirm which one of their models of DNA was

correct.

Photograph of DNA taken by Rosalind Franklin

Today, X-ray crystallography serves its purpose in many laboratories throughout

the world. It is primarily used for purposes that necessitates observing the structure of a

certain molecule or protein. X-ray crystallography provides a detailed three dimensional

image of the object being observed. Scientists may also use X-ray crystallography to

take samples of proteins and see how their conformation may have changed before and

after exposure to certain environmental factors. One would take an image of a protein

before the exposure, and then take another image after the exposure and see if the protein

denatured and or formed different secondary or tertiary structures. Of course the proteins

would not be the same exact protein, one would have to use a number of the same kind of

protein and select one to be X-rayed before the experiment and one to be X-rayed after


6/9

the experiment. It should be noted that X-ray crystallography, however, is not a very

good method for observing this type of change, it will be discussed in more detail in the

next section.

Difficulties with X-ray Crystallography

X-ray crystallography is a great way to observe proteins and protein structure but

the requirements for viewing are a bit demanding. It is necessary for one to crystallize

the protein before shooting X-rays at it. This will in it of itself render the protein

unusable. Therefore, one needs to make sure that the sample being observed is not the

only sample they have if the protein is needed for other processes. The actual

crystallization of the substance (atom or protein) poses its own dilemmas. One needs to

consider different factors that may distort the crystallization of the protein. These factors

include the size, the shape, the salinity, the charge, and other factors the macromolecule

(protein).

Another difficulty, one which many may overlook until the task is actually upon

them, is obtaining the pure protein to crystallize to begin with. There are several ways to

purify protein; different types of column chromatography are popular ways of doing so.

In column chromatography, proteins are separated based on factors similar to the ones

described above, i.e. shape, size, charge, etc. Column chromatographies are done in

cylindrical glass columns that are filled with permeable support media (beads). The

support media serves the purpose of impeding the flow of a solution with a mixture of

proteins that you may or may not want, and selecting for the protein wanted. There are

generally three types of column chromatography. One is ion exchange chromatography,

which separates molecules based on different charges. Another is size exclusion


7/9

chromatography, which as the name states, separates based on the size of the molecule.

The last one being mentioned is affinity chromatography, which separates based on

biological activity. This refers to the affinity of the protein to bind to certain ligand (a

ligand is an ion, a molecule, or a molecular group that binds to another chemical entity).

This means that the separation media will separate based on the molecules affinity in the

solution to bind to the antibodies on the separation media. Below are illustrations of the

above mentioned column chromatography methods.

Ion exchange Chromatography Size Exclusion Chromatography Affinity Chromatography

The arguably biggest problem with X-ray crystallography is the fact that the

molecule needs to be crystallized. Because of this, as stated in the section before this

one, the molecule itself will be unusable or at least drastically altered after crystallization

that it cannot be used again. Therefore, it is not the best way to observe change of

conformation of protein and it is impossible to do in vivo.

Conclusion

X-ray crystallography is a great way to observe objects that one wants to understand the

structure of. It can offer a clear, three dimensional, and detailed image of how a protein


8/9

is built and what secondary and tertiary structures exist within the macromolecule. It is

extremely useful if one does not know much information about a certain protein and

wants to know its characteristics. Using X-ray crystallography, one can determine the

proteins structure and therefore many of the characteristics that the protein has. Through

observing the structure of a protein, one will be able to see what the molecule may or

may not be attracted to and how it would react and function in certain solutions.

Different structures may show whether the molecule is hydrophobic or hydrophilic,

whether the molecule will bind with other molecules, and much more.

X-ray crystallography can be used to observe change but only in way that a

camera can take snapshots of something that may be better be recorded by a camcorder.

If one were to use X-ray crystallography to observe change, he or she would take images

of a single molecule from a sample before and after that sample is exposed to some type

of factor that may or may not change the conformation of the molecule. There are better

tools, possibly such innovative tools as confocal microscopes, which can be used to shoot

video without physically changing its samples, to observe change. X-ray crystallography

requires that the molecule being observed to be crystallized, which makes the molecule

unable to perform its normal function.

X-ray crystallography is an excellent tool for observing atom or molecule

structure, currently there may be no other tools that do it better. Its can be used to

observe change but there are better and more apt tools to fulfill that need.

References


9/9

1. Chemical Heritage Foundation.

http://www.chemheritage.org/classroom/chemach/pharmaceuticals/watson-

crick.html. 2005

2. Gaidos, Susan. http://news.uns.purdue.edu/html4ever/9804.Crystallography.html.

Purdue. 2006

3. Carter, Brandon and Robin L. Carter.

http://crystal.uah.edu/~carter/protein/xray.htm. 2000

4. http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Proteomics_and_Pr

otein_Expr/Protein_Analysis/XRay_Crystallography/Crystallization_Plates.html#

Greiner%20CrystalQuick%20Sitting%20Drop%20Protein%20Crystallization

%20Plates. 2006

5. http://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htm. 2006

6. Miller, Russ.http://www.cse.buffalo.edu/faculty/miller/Talks_HTML/CS501-

98/Part1/sld001.htm. 2006

7. King, Michael W. http://www.indstate.edu/thcme/mwking/hemoglobin-

myoglobin.html. IU School of Medicine. 2006
http://www.chemheritage.org/classroom/chemach/pharmaceuticals/watson-crick.htmlhttp://www.chemheritage.org/classroom/chemach/pharmaceuticals/watson-crick.htmlhttp://www.chemheritage.org/classroom/chemach/pharmaceuticals/watson-crick.htmlhttp://crystal.uah.edu/~carter/protein/xray.htmhttp://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htmhttp://www.cse.buffalo.edu/faculty/miller/Talks_HTML/CS501-98/Part1/sld001.htmhttp://www.cse.buffalo.edu/faculty/miller/Talks_HTML/CS501-98/Part1/sld001.htmhttp://www.cse.buffalo.edu/faculty/miller/Talks_HTML/CS501-98/Part1/sld001.htmhttp://www.indstate.edu/thcme/mwking/hemoglobin-myoglobin.htmlhttp://www.indstate.edu/thcme/mwking/hemoglobin-myoglobin.htmlhttp://www.chemheritage.org/classroom/chemach/pharmaceuticals/watson-crick.htmlhttp://www.chemheritage.org/classroom/chemach/pharmaceuticals/watson-crick.htmlhttp://crystal.uah.edu/~carter/protein/xray.htmhttp://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htmhttp://www.cse.buffalo.edu/faculty/miller/Talks_HTML/CS501-98/Part1/sld001.htmhttp://www.cse.buffalo.edu/faculty/miller/Talks_HTML/CS501-98/Part1/sld001.htmhttp://www.indstate.edu/thcme/mwking/hemoglobin-myoglobin.htmlhttp://www.indstate.edu/thcme/mwking/hemoglobin-myoglobin.html

Andrew Chen Xraycrystallography

Documents

Transcript of Andrew Chen Xraycrystallography