Protein Crystallography
Mitchell Guss
University of Sydney
Australia

Outline of the talk
• Recap some basic crystallography and history• Highlight the special requirements for protein(macromolecular) structure determination• Emphasize the role played by synchrotron radiation• Introduce some of the future challenges andopportunities offered by newly emerging
technologies

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100+ years of X‐ray crystallography• 1912 Max vonLaue and hiscolleagues
demonstrate thata crystal (coppersulfate) candiffract X‐rays• Awarded theNobel Prize 1914

Friedrich, Knipping and von Laue, 1912• Showed that X‐rays behaved as waves• Failed to correctly relate the scattering to theunderlying structure

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100+ years of X‐ray crystallography••1912 William Lawrence
Bragg interpreted thediffraction from sodium
chlorideAwarded the Nobel Prize
with his father (W.H.
Bragg) in 1915
– W.L. Bragg still remains theyoungest science Nobel
laureate at 25 years of age

Braggs, 1912
• Showed that the diffraction was equivalent to thereflection from planes drawn through the crystal• Could predict the positions of the diffracted spots
using Bragg’s law: λ = 2dsinθ• Solved many inorganic structure (e.g. NaCl) in thenext few years
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Timeline of biological crystallography ‐ 1•
•••
•
••
•

1895
1912
1912
1929
1951

Röngten: discovers X‐rays (Nobel 1901)
von Laue: demonstrates diffraction (Nobel 1914)
Braggs: first crystal structure (Nobel 1915)
Sumner: crystallizes an enzyme (Nobel 1946)
Pauling: protein components α‐helix and β‐sheet(Nobel Chemistry 1954, Peace 1962)
1953 Watson, Crick & Wilkins: DNA structure (Nobel 1962)
1954 Crowfoot‐Hodgkin: vitamin B12, penicillin(Nobel 1964)1959 Kendrew & Perutz: myoglobin and haemoglobin(Nobel 1962)

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Timeline of biological crystallography ‐ 2••••••

1970+ Klug crystallographic electron microscopy and nucleic
acid‐protein complexes (Nobel 1982)
1985 Michel, Deisenhofer & Huber: the photoreaction
centre, first membrane protein (Nobel 1988)
1997 Boyer & Walker: ATPsynthase a rotating molecule(Nobel 1998)
1998 MacKinnon: K+ ion channel (Nobel 2003)
2000 Yonath, Ramakrishnan & Steitz: the ribosome (Nobel2009)2011 Lefkowitz & Kobilka: G‐protein coupled receptors(Nobel 2012)

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The beginnings of (X‐ray) protein crystallography• 1930’s Dorothy Crowfoot (later Hodgkin) and J.D.
Bernal made the first X‐ray diffraction pictures of anenzyme – they realised that the crystals had to bekept wet or they didn’t diffract• 1937 Max Perutz began the structure analysis of
haemoglobin with W.L. Bragg as his supervisor – thestructure was not solved until 1961!
What has changed from 1937 to today that enables thesame structure to be solved in minutes?

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Excellent text for biomolecular crystallography• Excellent coverage ofboth the theory andpractice of the subject• Many of my illustrations
come from this text
• See Bernard Rupp’s
website for more detail
http://www.ruppweb.org/default.htm

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What is a crystal?
• An solid containing ofatoms or molecules that
repeats in threedimensions• Or, the convolution of a
motif (molecule) with alattice
Convolution• Place an instance of the motif at eachpoint of the lattice

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In this case a “duck” is convoluted
with a 2‐dimensional lattice to give a
2D “duck crystal”
93‐dimensional protein crystals• The unit lattice is the 3D
set of points described by
the 3 non‐collinear
vectors that give therepeating units• The motif is the contentof the unit lattice (Note: it
may be one or a numberof actual molecules)• The unit cell is the unit
lattice with its contents• The crystal is the 3D arrayof unit cells that fill space Not true for all systems anymore. Why?
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What is diffraction?
••

Diffraction takes place with sound;with electromagnetic radiation, suchas light, X‐rays, and gamma rays; andwith very small moving particles suchas atoms, neutrons, and electrons,
which show wavelike properties.
The phenomenon is the resultof interference (i.e., when waves aresuperposed, they may reinforce orcancel each other out) and is mostpronounced when the wavelength of
the radiation is comparable to thelinear dimensions of the obstacle.
Since X‐rays are of approximately thesame wavelength as inter‐atomic
bonds they will be diffracted bymolecules and crystals.

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Scattering from a molecule

The image is the scattering of the 2D projectionprotein molecule on the left. It is the sum of thescattering from each atom (j at position S). The
diffraction (FS) is continuous and can occur in any
direction S.
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Diffraction from a lattice(a, b) Diffraction from a line ofevenly spaced dots (or holes) is aseries of lines perpendicular to therow of dots. The lines have inverse
spacing to the dots.
(c) Diffraction from a 2D array ofdots is an array of dots at rightangles to the original rows.
The array of dots in (c) is theconvolution of the dots in (a) and(b). The diffraction pattern in (c) isthe product of the diffraction
patterns in (a) and (b). What you
see in the (c) is a real lattice and theassociated reciprocal lattice

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The convolution theorem & diffraction from a crystal

The convolution theorem states that undersuitable conditions the Fourier transform of a
convolution of two functions is the point wise
product of Fourier transforms of the individualfunctions.
Remember a “crystal” is the convolution of alattice and a “molecule”. Therefore thediffraction from a crystal will be the product ofthe diffraction of the lattice and of onemolecule.

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Scattering from a protein crystal

The effect of the crystal is to reinforce the diffraction signal at points where the waves from all
the molecules are in phase. This makes the signal sufficiently large to be measured but all theinformation is theoretically in the scattering from one molecule.

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The so‐called ‘phase problem’I(hkl) ∝ |F(hkl)|2
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The crystallography experiment

The X‐ray crystallography experiment is like a light microscope except (1) use X‐rays
and not visible light and (2) we have no objective lens for X‐rays so we cannot
refocus to make the image. In other words while we can measure the amplitudesof the diffracted waves we cannot measure their relative phases. This is the“phase problem” of crystallography.
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What is so special about protein crystallography?
• Asymmetric unit contains more atoms– Even a small protein (50 aa residues) has about 500 non‐Hatoms. A large protein may of many thousands• The unit cells are generally larger (V > 125,000Å3)– Diffraction is weaker (Ihkl  1/Vc)• Crystals diffract to lower resolution– Poorly ordered typically 30 – 80% solvent by volume• Combination of lower resolution, large atom
numbers and non‐centric space groups– direct methods for structure solution applicable to small
molecules do no work for proteins
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Overview of protein structure determination

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Life wasn’t meant to be easy!

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Importance of data quality (resolution)

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What has been achieved since 1962

Protein Data Bank (PDB) depositions by year
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PDB depositions by technique

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The experiment
1.
2.
3.
4.
5.
6.
7.
8.

Expression of proteins using molecular biology instead of isolationfrom natural sources – makes more and most importantly purer
protein
Grow crystals – the most uncertain and often the most time
consuming step – use of factorial screens and robotics
Record diffraction data – high speed detectors and intense
synchrotron sources can reduce what once took months tominutes or even seconds
Solve the structure – determine the phases. New methods
including anomalous phasing have significantly improved this step
although it can still be difficult in some cases
Build a model – aided by highly intelligent computer graphics
Refine the structure – optimise the model to fit the data. Fastcomputers have reduced this step from hours to seconds
Deposit the structure in the Protein Data Bank, and;Write a paper – often the slowest step

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CRYSTALLISATION

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Vapour diffusion – in theory!

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Different apparatus

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Crystallisation plates – for vapour diffusion

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What you are looking for!

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The basic crystallisation diagram

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Successful vapour diffusion experiment

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Screening – can’t do everything

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Can you predict whether a protein will crystallise
The xtalpred server: http://ffas.burnham.org/XtalPred‐cgi/xtal.pl“More efficient to improve the inherent crystallisability than to do furtherscreening” Rupp, 2010
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DATA COLLECTION (SOURCES)

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Laboratory X‐ray sources

Beam size at the sample 100 ‐ 200μ depending on optics

In the 1960s and 1970s typicalproblems were considered
insoluble unless crystals at least
300μ could be grown. Even withmodern optics most protein
crystals suitable for a laboratory
source are > 100μ.

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Synchrotron radiation and protein crystallography• Brilliance, small beam
– Use of small crystals; rapid data collection• Low Divergence– Resolve close reflections from large unit cells e.g. viruses• Tunability– Enhance anomalous signal for phasing• Pulsed beam
– Perform time‐resolved experiments on enzymes

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Biologists were not always welcome at synchrotrons

Synchrotron

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Protein crystallography at the Australian Synchrotron• Two beamlines
1) Bending magnet, highthroughput, MADphasing, small
moleculecrystallography2) Undulator, highbrilliance, small crystals• Extensive use of
remote access– large country, only one
synchrotron
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Protein crystallography endstations• Robot• CCD detector– Would like Dectris!

• Overhead frame– Easy access– But less stable• On‐line software– Processing on‐the‐flyallows for rapid
evaluation of crystals

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DATA COLLECTION (DETECTORS)Cheiron 201540

Detectors ‐ film
• Film– Cheap and linear
response – no longer
used except for teaching– Requires use ofdarkroom– Requires separate
scanner to digitize theimages– Can provide overall viewillustrating the crystal
symmetry

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Precession camera and image

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Detectors – image plates•

Image plates– Essentially a reusable
plastic film
• Read out by laser that
detects excited centres
created by X‐rays• Erased by light– Relatively cheap forlarge area. Widedynamic range. Low
noise.
– Relatively slow readout(about 120 s).
• Restricts usefulness at asynchrotron where anexposure is about 1s.
• Still used at homesources whereexposures are 1‐10minutes

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Detectors ‐ CCD• CCD– CCD. Need to be tiled forlarge area. Requires
cooling to reduce noise.
Relatively fast readout (<1s). Need to demagnify
the image to match CCDsize.
– Relatively expensive.
Need careful calibration.
– Physically large

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ADSC CCD detector

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Detectors – pixel arrays• Pixel arrays– Can tile manymodules.
– Virtually zeronoise.
– Operates atroomtemperature.
– Ultrafast readout
permits
continuousexposure.
– Very expensive –early adoption of
new technology.

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STRUCTURE SOLUTION

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Solving the phase problem••••

Marker atom substructure methods do not depend on prior structural knowledge
about the protein (other than the sequence and the structures of the aminoacids). Once the marker atoms (heavy metals, selenium) have been located byPatterson or direct methods starting phases for the modelling can be calculated.
These are de novo or experimental phasing methods.
Density modification are powerful methods for improving the initial phases andare used in practically all de novo phasing experiments. These methods included
solvent flattening, and non‐crystallographic symmetry averaging. As they do not
require any model information they may also be considered experimentaltechniques.
Molecular replacement require a similar structure as a molecular search probe.
Replacement is to be understood as the placing of the search model in the crystal
structure not as “substitution”. Phase bias can be a serious problem with thistechnique.
Direct methods exploit the fact that relationships exist between certain sets ofstructure factors. They require very high resolution (1.2 Å or better) for ab initio
determination of a protein structure and have been limited to very small proteins.
They are commonly used for small molecule structures. They are, however, very
important in sub‐structure (marker atom) determination.

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The concept of isomorphous difference
If we add an atom to a protein
crystal and change nothingelse (isomorphous) then thedifference in the diffraction
patterns of the “derivative”
structure with added heavy
atom and the “native” protein
structure approximates thediffraction pattern from theheavy atom alone.

FPA = FP + FA and thus
FA = FP − FA but these are vectors and the amplitudes (things we can measure) don’t add
up: FA ≠ FPA − FP but we can still use the differences to solve the sub‐structure.
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Revision – graphical construction of the structure factor

Graphical illustration of thesummation that yields one
structure factor for a 7‐atomstructure – with 6 relatively
light atoms and one heavier
atom. The resultant structure
factor is Fh.
atoms

Fh   f hj0 .exp(2ihx j )j 1

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The “true” isomorphous differences
When computing the totalstructure factor thecontributions from theindividual atoms are
summed vectorially. Thus, if
we add one more atom to aprotein we simply add its
structure factor to that of
the total for the protein.
The radii of the circles are
the amplitudes for theprotein and heavy‐atom
derivative structure factors.

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Graphical illustration of phase determination
Once we know where theheavy atom is we cancalculate FA – the amplitude
AND phase of its contribution.
For each reflection we canmake the construct closing
the vector triangle. We know
the amplitudes FP and FPA
from the experimental
measurements and the total
contribution FA.
If we draw a circle radius, FPA,
at the origin and then movealong FA and draw a circle
radius, FP, there should be
TWO points of intersectionthat satisfy the phase addition
giving two solutions for φP.
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Resolving the phase ambiguity• We have a phase ambiguity if there is: a single
isomorphous derivative (SIR) or anomalous scatteringfrom the native crystal (e.g. from sulfur) (SAD).
This problem can be resolved:• By adding more derivatives – multiple isomorphous
replacement (MIR)• By using the anomalous signal – single isomorphous
replacement with anomalous scattering (SIRAS) or
multiple isomorphous replacement with anomalous
scattering (MIRAS)• By using the dispersive and anomalous signal together –multiple wavelength anomalous diffraction (MAD)• With the help of density modification• With dedicated direct methods programs
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Anomalous scatteringf(S, )  f  f(  )  f0(S )'"( )The scattering from an atom actually contains two wavelength (energy) dependent
terms. These vary rapidly near an absorption edge.
f(S, )  f(S0 )  f(' )  f(" )Cheiron 201552

Anomalous scattering for phasing

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Anomalous scattering only significant for heavier elements in a protein• At most synchrotrons PX
beamlines one can accessthe L‐edges for elements
form iodine to uranium
and the K‐edges fortransition metals• Most importantly theedge for selenium is at0.979 Å (1.27 KeV)
– Se can be introduced into
recombinantly expressed
proteins replacing thesulfur in the amino acidmethionine

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Single Isomorphous Replacement with Anomalous Scattering(SIRAS)In this case for eachreflection we have 1
observation of thenative amplitude, FP,
and two for thederivative, FPA‐ and FPA‐.
In the absence of
anomalous scattering(blue circle) there are
two solutions withanomalous scattering(light green circles)
there is only one.

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Summary of structure solution methods

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