X-ray Crystallography

X-ray Crystallography

Method for studying the three

dimensional, atomic structure

of molecules (protein).
 Introduction to:
X-Ray Crystallography
• x-rays are used to probe the
protein structure
• Crystals are used because it
helps amplify the diffraction
signal.
• xrays interact with the
electrons surrounding the
molecule and “reflect”. The
way they are reflected will be
prescribed by the orientation
of the electronic distribution.
• What is really being
measured?
– Electron Density!!!
A protein crystal

is placed in the x-ray beam

the x-rays are

diffracted by the
electron clouds
around atoms
the atomic structure can
be deduced from the
data
 Crystals

• The ideal crystal

• The real crystal

• The protein crystal

 The Ideal Crystal

• The ordered disposition of molecules such

that there exists a regular repetition of a
pattern in 3-D space, where this repetition
extends over a distance equal to or greater
than thousands of molecular dimensions.
 The Real Crystal

• A crystal with less than perfect

periodicity, imperfections are often
caused by impurities and the effects of
non-zero temperatures.
 The Protein Crystal

• The crystal contains a high degree of

solvent, meaning that some molecules
present are not in the crystalline state,
but in the liquid state, creating disorder.
 Experimental Set- Up
Cryostream

Beam Stop Rigaku rotating copper anode

(in-house source)
Detector Monochromator
Crystal Or Mirrors

X-ray source

X-ray beam

Goniometer
 How are X-rays produced?

• X-rays in the useful range for crystallography

(around 1 Å) can be produced by bombarding
a metal target (most commonly copper or
molybdenum) with electrons produced by a
heated filament and accelerated by an electric
field.
• A high energy electron collides with and
displaces an electron from a low lying orbital in
a target metal atom.
• Then an electron from a higher orbital drops
into the resulting vacancy, emitting its excess
energy as an X-ray photon.
 X-ray Generators - The Rotating Anode

Rigaku
rotating
copper
anode
(in-house
source)

X-rays are generated by bombarding a rotating copper

anode with electrons. This creates X-ray radiation consisting
of two wavelenghts characteristic of copper sources, 1.54 Å
(K radiation) and 1.39 Å (K radiation). Crystallographers
usually use K radiation (the intensity is greater).
The oscillation equipment
Rotates the crystal about an axis () perpendicular to the
x-ray beam (and normal to the goniometer). The diffraction
pattern from a crystal is a 3-D pattern, and the crystal must
be rotated in order to observe all the diffraction spots.
 Detectors

1- Photographic film
Not much used anymore because
of the availability of far more
sensitive detectors. Superior
resolution due to its fine grain, but
limited dynamic range.
 Image plates
• Image plates are coated with a layer of inorganic storage
phosphor.
• X-ray photons excite electrons in the material to higher
energy levels.
• Part of the energy is emitted as fluorescence, but an
appreciable amount of energy is retained in the material.
• The stored energy is released upon illumination
with a red laser.
• Blue light is emitted and measured with a
photomultiplier. The light emitted is proportional
to the number of photons. Ten times more
sensitive than film, dynamic range (1:104-105)
 Bragg’s Law

• Bragg's law, or Wulff–Bragg's condition,

a special case of Laue diffraction, gives
the angles for coherent and incoherent
scattering from a crystal lattice.
• When X-rays are incident on an atom, they make
the electronic cloud move as does any
electromagnetic wave.
• The movement of these charges re-radiates
waves with the same frequency, blurred slightly
due to a variety of effects; this phenomenon is
known as Rayleigh scattering (or elastic
scattering).
 Rayleigh Scattering

• Named after the British physicist Lord Rayleigh

• It occurs when the size of the particle

responsible for scattering event is much
smaller than the wavelength of incident light
 Sky appearing blue
• Sun has red and blue lights that are scattered by molecules
in the air
• Light moves in wavelengths, some are long and some short

• Red, yellow and orange light waves are short so they do not
show through as much as blue
• The molecules scatter the blue light rays in different
directions
Bragg’s Law equation
2d sin = n
where
 = wavelength of incident x-rays
 = angle of incidence
d = lattice spacing
n = integer
Spots are observed when the following conditions are met:
1. The angle of incidence = angle of scattering.
2. The spacing between lattice planes is equal to
an integer number of wavelengths.
 X-Ray Scattering from a Crystal
A typical image of
x-rays scattered by
a crystal:

(Dark spots
are the
scattered x-
rays)
X-Ray Diffraction Pattern
Photographs of Jerome
Karle (left) and Herb
Hauptman (right), who
won the Nobel Prize for
their work on solving
the phase problem for
small molecule crystals.
 Applications

• Widely used in chemistry and

biochemistry to determine the structures
of immense variety of molecules
including organic compounds DNA and
proteins
• Determines electron density, the mean position
of atoms in the crystal, their chemical bond,
their disorder and various other information
• Characterizing the atomic structure of new
materials
 Factors that affect crystallization
1) Purity of proteins

2) Protein concentration

3) Starting conditions (make-up of the protein solution)

4) Precipitating agent (precipitant)

5) Temperature

6) pH

7) Additives: Detergents, reducing agents, substrates, co-

factors, etc.
 X-Ray Powder Diffraction (XRPD)
• More appropriately called polycrystalline X-ray diffraction, because it can
also be used for sintered samples, metal foils, coatings and films, finished
parts, etc.
• Used to determine:
– phase composition (commonly called phase ID)- what phases are present?
– quantitative phase analysis- how much of each phase is present?
– unit cell lattice parameters
– crystal structure
– average crystallite size of nanocrystalline samples
– crystallite microstrain
– texture
– residual stress (really residual strain)
– in-situ diffraction (from 11 K to 1200C in air, vacuum, or inert gas)