The excitement of building a structure cannot be exagerated. Until that point, the molecule is a black box; you know it exists and you know what it does, but not a lot more. Now, suddenly, it is as though the curtains have parted, and you see the molecule in its full glory, all of its atoms in their place revealing the twists and turns of the chain as it folds up into its unique architecture to show how it might work. It must be how explorers felt when they came across a completely new landscape.
Venki Ramakrishnan, Gene Machine
I’m currently reading The Code Breaker, a riveting account of the life and work of Jennifer Doudna - recipient of the 2020 Nobel Prize in Chemistry - by Walter Isaacson. Dr. Doudna’s early research involved determining the structure of the RNA, using techniques such as X-ray crystallography. This post is my attempt to understand and explain this important scientific technique in the simplest manner possible.
Stephen Curry, a structural biologist at the Imperial College in UK, explains X-ray crystallography beautifully in this Youtube video.
Why is it important to understand the structure of a molecule?
X-Ray crystallography is used to understand the three-dimensional structure of small and large molecules. Determining the structure of a molecule is important in understanding how it interacts with other molecules around it. For instance, the shape of your hands allows you to shake hands with others. If hands were shaped like feet, handshakes likely wouldn’t exist.
Why use X-rays?
Visible light only allows you to see the surface of things, you cannot see inside them. Visible light has large wavelengths. Atoms (the units that make up molecules) are orders of magnitude smaller than objects we see around us - we have billions of atoms in our bodies. Trying to view atoms with visible light is like trying to catch fish eggs with a net meant to capture whales - they’ll flow right past without being affected. X-rays are a kind of radiation with very small wavelengths. The wavelength of X-rays is about the size of an atom and so an X-ray is reflected off the atom when it strikes.
What is a crystal and why do we need them?
A single molecule is usually very small for there to be sufficient information in its diffraction pattern to determine the structure of the molecule. A group of several similar molecules is needed and this ‘group’ takes the form of a crystal in X-ray crystalloraphy.
A crystal is a structure in which molecules of the same kind arrange themselves in an ordered pattern. In a solution, molecules may be oriented in many different ways (like blocks in tetris), however in crystals, the molecules orient themselves in a specific direction (soldiers in a marching contingent). This regular orientation, helps the crystallographer to determine the placement of atoms in the molecule, by studying not just a single molecule, but several of them.
What happens in X-ray crystallography?
X-rays hit the crystal from different angles and the resulting scattering of X-rays (called diffraction) is captured to then understand the molecule structure. Molecules are made up of atoms. Atoms are made up of protons, neutrons and electrons. Electrons occur outside the nucleus of the atom. When an X-ray hits an electron, it causes the electron to oscillate and itself emit X-rays in all directions. However, the combined intensity of the X-rays emitted by the electron is slightly lesser than the intensity of the X-ray wave that struck it. The more electrons are present in a particular region of the molecule, the more the difference in intensity of light that struck it and the light reflected (or diffracted). Thus, the areas with high ‘electron density’ will leave more intense spots on the X-ray film. High electron density indicates the presence of atoms in that region of the crystal. With help from the angles at which X-rays strike the atoms, the angles at which the X-rays are reflected off, the intensity of the X-rays reflected and more data, scientists are able to determine the structure of the molecule.
Why is the intensity of the spot on the X-ray film important?
The atomic number of a element (such as hydrogen, carbon, iron) is the number of protons in its nucleus. This is also equivalent to the number of electrons the atom has. Atoms with more electrons (those known to have higher atomic numbers) will diffract X-rays more and appear as denser spots on the X-ray film. Our skin and tissues are primarily made of molecules containing carbon, oxygen, nitrogen, hydrogen - all elements with low atomic numbers. Bones contain Calcium, phosphate and other minerals with atoms having more electrons. This is why in an X-ray of your hand you see bones clearly, but the skin and tissues around them are diffuse and nearly transparent. Simply put, the intense, near-opaque spots on an X-ray film indicate the presence of atoms with more electrons. (Metals have very high atomic numbers compared to the atoms in the human body - hence, you’re required to take off metallic objects before going for a medical x-ray.)
Role of crystallography in understanding human biology:
Molecules in the body - biological molecules - are much larger and complex than the ones of salts or ores. They could contain several hundred atoms and are present in the form of intricate structures involving chains of atoms folded one on top of the other. (Imagine several hundred wired earphones all jumbled up together). The folding of the molecule is crucial for its function - incorrect folds can make the molecule completely incapable of performing its necessary biological functions. So it is important not just to know which atoms are present in the molecule, but also how and where and in which order they are arranged. Crystallography makes all of this possible and has played a major role in helping understand the CRISPR system which is now the forerunner in gene editing technologies.
Cooper Fun Fact:
The Curiosity Rover sent to Mars on 2021 has an instrument that can perform X-ray diffraction on materials collected from the planet!
References:
Seeing Things in a Different Light, video featuring Stephen Curry
Understanding Crystallography Part 1 and Part 2 by the Royal Institution