Protein Crystallography
Veronika Harmat
ELTE Institute of Chemistry, Laboratory of Structural Chemistry and Biology, ELTE-CrystalLAB,
Slides of the lectures, pdf format:
http://harmatv.web.elte.hu/Fehkr-segedanyag/
(user: szimmetria, password: P21212)
More detailed material in Hungarian:
http://harmatv.web.elte.hu/Rontgendiffrakcio-jegyzet/Rontgendiffrakc-szerk-vizsg.pdf
1) Introduction
Topics
• Historical background
• Basic concepts:
• The diffraction phenomenon; structure factor- Fourier synthesis
• Crystal symmetry
• Steps for structure solution:
• Crystallization, data collection
• Solving the phase problem: isomorphous replacement,
anomalous dispersion,
molecular replacement
• Model building, refinement
• Validation
1) Introduction
• New directions and hot topics in protein crystallography
X-ray crystallography - introduction
• Elastic interaction between X-ray radiation and the sample
• Interference between the scattered beam components
• Analogy with the microscope – a detailed, magnified image of the sample
C T S D O K
Ft
1) Introduction
But: it is not possible to make suitable quality lenses, so we can detect only the
diffraction image
X-ray crystallography - introduction
„Crystallography” – the highest information content is gained from diffraction
images of single crystals .
Single crystal: the atoms are continuously periodically ordered in three dimensions
Crystal structure of GFP 1) Introduction
X-ray crystallography - introduction
The detailed, magnified image of the molecules in
the crystal:
The interactions occurs between the
electromagnetic radiation and the electron cloud
of the molecules, so it is the electron density map
that can be calculated from the diffraction data.
So a model building step is required: atoms are
placed in the maxima of the electron density map.
1) Introduction
X-ray crystallography - introduction
Crystallographic phase problem: the phase information of the diffracted beams is
lost during the measurement, only the amplitudes can be directly calculated.
As a consequence the electron density Phase wavelength amplitude
maps cannot be calculated at this stage.
E.g. diffracting X-rays by three atoms
Incident beam
1) Introduction
X-ray crystallography - introduction
Crystallographic phase problem: the phase information of the diffracted beams is
lost during the measurement, only the amplitudes can be directly calculated.
As a consequence the electron density
Phase wavelength amplitude
maps cannot be calculated at this stage.
Incident beam
Positive or negative
interference depends on the
direction
1) Introduction
Information content of the dataset: resolution
Small molecules: atomic resolution(0.8-1Å).
Non hydrogen atoms can be individullly
placed
Macromolecules:
Maximal resolution is typically 1.5-3Å.
The lower resolution results less detailed
electron density maps (the atoms do not
have individual electron density maxima).
For describing molecular geometry data
from other resources are also needed
(geometric restraints).
Resolution:
1.2 Å 1.8 Å 2.8 Å 4.0 Å
1) Introduction
Steps of macromolecular crystallography
Crystallization
Data collection
Solving the phase
problem
Refinement
Validation,
interpreting
the structure
Model building 1) Introduction
Significance of high flux sources (synchrotron, XFEL) in macromolecular crystallography
Data collection method
X-ray All
All X-ray structur Structures in the Protein Data Bank
diffraction, www.rcsb.org
diffracti es
synchrotron
on
radiation 2020. March: 162043 structures,
1985- 88,9% X-ray diffraction, 8,0% NMR, 2,9%
3 187 191 CryoEM
1990
1991-
48 2168 2518
1995
1996-
5389 6674 8133
2000
2001-
11725 15316 17936
2005
2006-
27859 31753 35560
2010
2011- 1) Introduction
36346 41645 44867
2015
2016-
36349 39609 44595
2019
Developments accelerating protein crystallographic studies
1. Developments in molecular biology and protein purification
New methods(automatisation) Protein production
Structural genomics Solving the phase
Se-Met derivatives of proteins problem
2. Automatization of crystallization
Crystallization robots,
Crystallization
miniaturisation
3. Low temperature data collection (routinely)
Radiation damage of crystals is decreasing Data collection
1) Introduction
Developments accelerating protein crystallographic studies
4. Synchrotron sources
High brilliance X-rays: higher resolution
Improved detectors: fast measurements, lower
noise
White X-rays: Laue diffraction images, molecular
movie Data collection
Automatisation: “FedEX crystallography”, robotics
Tunable wavelength: use of anomalous dispersion Solving the phase
problem
5. User friendly and robust softwares
Automated data reduction, strucutre
determination: for good resolution strucutres
also model building Data collection, Structure 1) Introduction
determinbation, Validation
New refinement algorithms
New methods for solving the phase problem
X-ray diffraction: advances and limitations
Single crystal is needed
• Proteins with stable conformations can be studied (globular proteins)
• The conformation in solution and in the crystal is usually similar
• No limitations on size
Structures of ribosome
(more than 120 structures)
30S subunit: ~35000 atoms,
50S subunit:~64000 atoms
1) Introduction
V. Ramakrishnan Cell 108, 557,2002
X-ray diffraction: advances and limitations
The electron density map shows the average electron density within the crystal
during the data collection period
► Only the most permanent conformations of a flexible region can be
identified on the electron density map
► The information content of the electron density map as well as the error of
atomic positions depend on the resolution.
Electron density of the tryptophan ring at various resolutions
1) Introduction
1.0 Å 1.8 Å 3.0 Å 4.0 Å
X-ray diffraction: advances and limitations
Stable (or metastable states of the protein can be studied) routinely.
To extend possibilities:
• Mutations (inactive form of an enzyme)
• Ligands (e.g. inhibitors, analogues of natural ligands)
• Changing the pH, temperature..
1) Introduction
Short story of crystallography introduced
with Nobel prices
• Wilhelm Conrad Röntgen 1901. Physics
discovery of X-rays
1) Introduction
www.nobelprize.org
Short story of crystallography
• Max. von Laue 1914. Physics
discovery of X-ray diffraction by crystals
Copper
sulphate
• W.H. Bragg és W.L. Bragg 1915. Physics
solving crystal structures using X-ray diffraction (NaCl)
1) Introduction
NaCl, KCl, LiF… ‘graphite’
naphtaline
Short story of crystallography
• Ch.G. Barkla 1917. Physics
characteristic X-ray radiation of the elements
I
1) Introduction
λ (nm)
Short story of crystallography
• J.B.Sumner, 1946 Chemistry
crystallization of enzymes
1) Introduction
Urease crystals
Short story of crystallography
• F.C.H Crick, J.D. Watson, M.H. Wilkins 1962. Physiology
Structure of DNA
The first X-ray diffraction
image (fiber diffraction) A and B configuration of DNA 1) Introduction
Short story of crystallography
• M.F. Perutz, J.C. Kendrew 1962. Chemistry
structures of globular proteins (haemoglobin and mioglobin))
Haemoglobin 5.5Å resolution
Mioglobin 2 Å resolution
1) Introduction
1.4 Å resolution electron density map
Short story of crystallography
• D.Crowfoot Hodgkin 1964. Chemistry
solving the structures of biologically important compounds
(vitamineB12, penicillin)
Cianocobalamine (B12 vitamine)
p-bromo-phenoxy-methyl-penicillin
1) Introduction
Short story of crystallography
• J.Karle, H.Hauptmann 1985. Chemistry
developing the direct methods for
solving the phase problem
Absolute configuration of
batrachotoxinin (Br derivative)
Four conformers of ciklohexaglicine (1963)
1) Introduction
Short story of crystallography
• R.Huber, J.Deisenhofer, H.Mitchel • P.Agre, R.MacKinnon
1988. Physiology 2003. Chemistry
structure of the first membrane protein water and ion channels
(photosynthesis reaction center)
(αβ)6 hexamer
top view
RC cofactor
system
1) Introduction
Short story of crystallography
• P.D.Boyer, J.E.Walker, J.C.Skou • Roger D. Kornberg
1997. Chemistry 2006. Chemistry
enzyme mechanism of ATP synthesis molecular basis of eukaryotic
transcription
1) Introduction
bovine F1-ATase α3β3γ subunit
RNA polymerase II
Short story of crystallography
• V.Ramakrishnan, T.A.Steitz, A.E. Yonath
2009. Chemistry
Structure and function of the ribosome
1) Introduction
Short story of crystallography
• Robert J. Lefkowitz, Brian K. Kobilka
2012. Chemistry G-protein coupled receptors
β2-adrenerg receptor
-Gs protein complex
1) Introduction
Short story of crystallography
The Nobel Prize in Chemistry 2013 was awarded jointly to Martin Karplus,
Michael Levitt and Arieh Warshel "for the development of multiscale models for
complex chemical systems."
1) Introduction
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