X-ray Diffraction

Obtaining images of
macromolecules

In order for an object to be visible under magnification, the

wavelength () of the light must be, roughly speaking, no
larger than the object.

Visible light (400-700 nm) cannot produce an image of protein molecules,

in which bonded atoms are about 1.5 apart (0.15 nm). Electromagnetic
radiation of this wavelength falls into the X-ray range.

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 X-rays
X-rays are just another form of electromagnetic radiation:

Electromagnetic Spectrum:
Visible
X-rays Ultraviolet Light Infrared Microwave Radio
Energy:
High Low

Frequency:
High Low

Wavelength:
Short Long
~1(=0.1nm) ~400nm

Resolving Power: (Ability to see detail)

High Atomic Low
Resolution
{Electrons (~500keV, as in electron microscope) are not a form of electromagnetic radiation,
but they still have wave-like character (deBroglie wavelength ~0.01).
Unlike photons (EM rad.), electrons are charged --> fry the specimen faster }
M.Rould02
 X-ray Crystallography

a method for studying the three-dimensional,

atomic structure of molecules. In this course we
will concentrate on applications for biological
macromolecules.
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
 Why cant we visualize molecules directly?

AsinglemoleculeisaveryweakscattererofXrays.Mostofthe
Xrayswillpassthroughthemoleculewithoutbeingdiffracted.The
diffractedraysaretooweaktobedetected.

Solution:Analyzingdiffractionfromcrystalsinsteadofsingle
molecules.Acrystalismadeofathreedimensionalrepeatofordered
molecules(1014)whosesignalsreinforceeachother.Theresulting
diffractedraysarestrongenoughtobedetected.

Unlikevisiblelight,Xrayscannotbefocusedbylenses.Therefractive
indexofXraysinallmaterialsisverycloseto1.0.

Solution:Useacomputertosimulateanimagereconstructinglens.
Inshort,thecomputerplaysthepartoftheobjectivelens,computing
theimageoftheobject,thendisplayingitonascreen.
SylvieDoubli2000
 The nature of crystals
Under certain circumstances, macromolecules (protein, DNA, RNA)
can form crystals. The resulting crystal is a three-dimensional array of
ordered molecules held together by noncovalent interactions.

Evidence that solution and crystal structure are similar:

1- NMR and X-ray crystallography have been used to determine the

structure of the same molecule. The two methods produce similar
models.
2-Many macromolecules are still functional
in the crystalline state.

Most protein crystals contain 50-70% solvent.

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 What is a Crystal ?
objectformedbystackingabasicunitinall3dimensions

Unit Cell

M.Rould02
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.
 Supersaturation
toaddmoreofasubstance(toasolution)thancannormallybe
dissolved.Thisisathermodynamically unstable state,
achieved most often in protein crystallography by vapor
diffusion or slow evaporation techniques.
Zone 1 - Metastable zone.
The solution may not nucleate for a long time
but this zone will sustain growth.
It is frequently necessary to add a seed crystal.

Zone 2 - Nucleation zone.

Protein crystals nucleate and grow.

Zone 3 - Precipitation zone.

Proteins do not nucleate but precipitate out
of solution.
Diagram from the website for The University of Reading, Course FS460
Investigating Protein Structure and Function
 Nucleation

phenomenon whereby a nucleus, such as a dust particle, a

tiny seed crystal, or commonly in protein crystallography,
a small protein aggregrate, starts a crystallization process.

Common difficulties:

1. If supersaturation is too high, too many nuclei form,

hence an overabundance of tiny crystals.

2. In supersaturated solutions that dont experience

spontaneous nucleation, crystal growth often only occurs
in the presence of added nuclei or seeds.
 Cessation of growth
Caused by the development of growth defects
or the approach of the solution to equilibrium.

Mother liquor
The solution in which the crystal exists - this
is often not the same as the original
crystallization screening solution, but is
instead the solution that exists after some
degree of vapor diffusion, equilibration
through dialysis, or evaporation.
 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.
Hanging/Sitting Drop Vapor Diffusion
Most popular method among protein
crystallographers.

1. Crystal screen buffer is the well solution

(0.5 - 1 mL)
2. Drop (on siliconized glass cover slip) is 1/2
protein solution, 1/2 crystal screen buffer (0.5-
4 L). So, the concentration of precipitant in
the drop is 1/2 the concentration in the well.
3. Cover slip is inverted over the top of the
well and sealed
with vacuum grease (airtight).
4. The precipitant concentration in the drop
will equilibrate with the precipitant
concentration in the well via vapor diffusion.
Interpreting the Results of the
Crystallization Experiment
 The Hampton Crystal Gallery
http://www.hamptonresearch.com/stuff/gallery.html
 Experimental Set- Up
Cryostream

BeamStop Rigaku rotating copper anode

(in-house source)
Detector Monochromator
Crystal OrMirrors

Xraysource

Xraybeam

European Synchrotron
S. Cates 02 Radiation Facility
Goniometer Grenoble, France
 How are X-rays produced?
X-rays in the useful range for crystallography (around 1 ) 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.

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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 (K radiation) and 1.39
(K radiation). Crystallographers usually use
K radiation (the intensity is greater).
X-ray Generators - The Synchrotron

European Synchrotron
Radiation Facility
Grenoble, France

Electrons (or positrons) are released from a particle accelerator into a storage ring.
The trajectory of the particles is determined by their energy and the local magnetic
field. Magnets of various types are used to manipulate the particle trajectory.
When the particle beam is bent by the magnets, the electrons (or positrons) are
accelerated toward the center of the ring. Charged particles moving under the
influence of an accelerating field emit electromagnetic radiation, and when they are
moving at close to relativistic speeds, the radiation emitted includes high energy x-
ray radiation.
 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.

Check out Bernhard Rupps Crystallography 101 website:

http://www-structure.llnl.gov/Xray/101index.html
 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.

2- 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 (1:10 4-105)

S.Doubli2000
 Diffraction
A characteristic of wave phenomena, where whenever
a wavefront encounters an obstruction that alters
the amplitude or phase of a part of the wavefront,
diffraction will occur.

The components of the wavefront, both the

unaffected and the altered, will interfere with one
another, causing an observable energy-density
distribution referred to as the diffraction pattern.
Interactions between X-rays and atoms

X-rays are scattered almost exclusively by the electrons

in the atoms, not by the nuclei.

The incident electromagnetic wave exerts a force on the

electrons. This causes the electrons to oscillate with the
same frequency as the incident radiation. The oscillating
electrons act as radiation scatterers and emit radiation
at the same frequency as the incident radiation.

S. Doubli 2000
When an incident x-ray beam hits a
scatterer, scattered x-rays are
emitted in all directions. Most of Destructive Interference
the scattering wavefronts are out
of phase interfere destructively.
Some sets of wavefronts are in
phase and interfere constructively.

A crystal is composed of many

repeating unit cells in 3-dimensions,
and therefore, acts like a 3-
dimensional diffraction grating. The Constructive Interference
constructive interference from a
diffracting crystal is observed as a
pattern of points on the detector.
The relative positions of these
points are related mathematically to
the crystals unit cell dimensions.
Diffraction gratings Diffraction patterns

Notice - when the diffraction grating gets smaller, the

pattern spacing gets larger (inverse relationship)
Braggs Law

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 When x-rays scatter
scattered x-rays) from a crystal we see
discrete spots:
Reflections

Why?

X-Ray Diffraction Pattern

M.Rould02
 X-Ray Diffraction from a Crystal
Electromagnetic radiation is wave-like:

Electric
Direction
+ + + + + +
field
of motion

- - - - - of x-ray
photon

Waves can add constructively or destructively:

Electric

+ + + + + +
field

- - - - -
Sum

M.Rould02
 Fourier Methods in Diffraction Theory
Foreachpointinadiffraction,thereisacorrespondingspatialfrequency.
Therefore,thedistributionofafarfielddiffractionpatternistheFourier
transformoftheaperturefunction.(apertureanopening,often
adjustable,thatcontrolstheamountoflightreachingthelensonacamera
orotheropticalinstrument.)

Inourcase,theaperturefunctionistheregularlyperiodic(duetothe
repetitionoftheunitcellinthelattice)electrondensitydistributionwithin
ourcrystals.TheelectrondensityistheinverseFouriertransformofthe
diffractionpatternexpressedasfollows:

(x,y,z)=1/VunitcellF(hkl)e
h k l
2i(hx+ky+lz)
,

whereVunitcell=volumeofoneunitcellandF(hkl)iscalledthestructure
factorforaparticularsetofMillerindicesh,kandl.Wecandoa
summationhere,insteadofintegrating,becauseweknowwewillonly
havereflectionsatintegervaluesforh,kandl.
 Electron Density

Electrondensitydistribution:
(x,y,z)=1/VunitcellF(hkl)e2i(hx+ky+lz)
h k l

forconvenience,letussubstitute=2(hx+ky+lz)inthefuture

TheamplitudeofthestructurefactorF(hkl)foranygiven
reflectionisproportionaltothesquarerootoftheintensityof
thediffractedbeam,or: |F(hkl)|2I

Therefore,wecandeduce|F(hkl)|,themagnitudeofF,directly
fromourdata,butnotitsphase.
R factor
Measure of the crystallographic residual, indicates
the correctness of a model:

R = | (|Fobs|-|Fcalc|) |
(|Fobs|
Variations that can prove confusing to the novice:

Rmerge measurement of the quality of a merged data set

Rsym measurement of the variation between symmetry-related

reflections

Rfree R factor for a test set of unique reflections that have

been omitted from the refinement process (unbiased)
 Positional Refinement
The atomic position parameters x, y and z are refined for
each atom.

Difficulties in protein crystallography:

large number of parameters to fit
macromolecular crystals diffract weakly, producing a
poor parameters to observations ratio.

The geometrical constraints introduced by the

conformational energy terms greatly reduces the number
of parameters to be refined. Least-squares optimization
or conjugate gradient minimization techniques are
commonly used for finding the best fit of the model to the
data.
B-factor (temperature factor) refinement
Bfactorsareindicatorsofatomicmobility.Highvalues
correspondtolowelectrondensity,indicatingadynamic
ordisorderedregion,orapossibleerrorinposition.

TheBfactorisanexponentialexpressionappliedtothe
scatteringfactorthatrelatestothethermalmotionofthe
scatteringatomandthedecreaseinscatteringintensitythat
resultsfromthermalmotions.

2 2
fe B[(sin)/]

Thexrayenergytermismodifiedinthetargetenergy
functionisrevisedwhereFcalcisreplacedbyFcalcesB/4
2

 Occupancy Refinement

The occupancy factor is used to describe disorder

in the model. An atom with a partial occupancy
factor can be thought of as an atom that does not
occupy that position 100% of the time (i.e., ions,
water, cofactors). Some refinement programs do
not require that the occupancy factor be 1, so it
is up to the crystallographer to remember that 1
is the upper limit on the occupance factor for a
given atom in a given position.
 Maps
Electrondensitydistribution:
(x,y,z)=1/VunitcellF(hkl)e
h k l

Thefirstmapisanapproximationtothetrueelectrondensity
derivedfromtheobservedstructurefactoramplitudes(Fobs)
andtheestimatedphasesfromthemodel(MR,MAD,orMIR
phases).

(Rememberourillustrationsthatthecorrectnessofthemodel
imagedependsmoreonhavingthecorrectphaseinformation
thanonhavingthecorrectamplitudes.)
 Maps contd
Both tryptophans are from the same 1.7 crystal structure,
but the map in Figure 1 is the first map calculated using the
initial MR phases and the map in Figure 2 is the final map
calculated using the refined phases.

1 2
 Resolution limits

6.0 - 4.5 Placement of secondary structures

3.0 Chain tracing
2.5 Side chain orientation
1.8 Alternate side chain orientations
1.2 Hydrogen atoms
 PDB Validation Suite
PROCHECK, NUCHECK, SFCHECK

PROCHECK
Assesses the geometry of the residues in a given protein structure,
as compared with stereochemical parameters derived from well-
refined, high-resolution structures.

Unusual regions highlighted by PROCHECK are not necessarily errors,

but may be unusual features for which there is a reasonable
explanation (eg distortions due to ligand-binding in the protein's
active site). Nevertheless, they are regions that should be checked
carefully.

The only input required for PROCHECK is the PDB file holding the
coordinates of the structure of interest.
Practical Considerations - generalizations (that means,
of course, that there are always exceptions)

Resolution: Good: 2
For sidechain conformations:
< 3

R-factor: Good upper limit for ~ 2 data: 20 - 23 %

R-free: within 10% of R
(closer for hi res)