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EuropeanMolecularBiologyLaboratory
Grenoble Outstation
Growing Biological crystals for Neutrons
Monika Budayova-Spano
UJF – EMBL – CNRS / ILL
Why neutron protein crystallography?
Providing evidence on the protonation state of the inhibitor and residues within the active site and on the solvent structure surrounding a protein which cannot be seen by X-ray analysis. Neutron diffraction can be used to directly determine the positions of H-isotopes at medium resolutions (~2.5 Ǻ).
The importance of H-atoms
Enzyme mechanisms; location of H-atoms aids our understanding of catalytic activity
Ligand binding interactions; identification of key hydrogen bonds
Solvent structure; can play an important role in some physiological and enzymatic functions
H/D exchange; identification of solvent accessible areas
Identifying the positions of hydrogen atoms in a structure helps us understand how the protein functions.
Hydrogen atoms mediate structure and function.
Vitamin B12
TrypsinEndothiapepsin
1:1 co-crystal of BPY and thiodiglycolic acid
Neutron protein crystallography
Advantages;H/D more readily visualized with
neutrons than with X-rays
Able to distinguish between hydrogen isotopes
Non-destructive probe – no radiation damage, thus can collect data at room temperature
Limitations:Low flux of neutron beams
Large sample size required
Long times scales
Only a few (<20) high-resolution neutron structures have been solved!
X-rays
Positions of all non-hydrogen atoms of a protein structure e.g. C, N, O, S.
High resolution X-ray data (better than 1.2 Å) required
…even then only those H-atoms which are extremely well localized can be seen.
X-rays
Scattered from electrons
Scattering proportional to ZH C N O Al Si P Ti D
1 6 7 8 13 14 15 22 1
-37.4 66.5 93.6 58.0 34.5 41.5 51.3 -34.4 66.7
NeutronsScattered by nuclei
Scattering not proportional to Z
H C N O Al Si P Ti D
1 6 7 8 13 14 15 22 1
•Neutron diffraction can be used to directly determine the positions of H-isotopes at medium resolutions (~2.5 Ǻ)
•Less variation between the elements•Large difference in the cross-section among isotopes
Neutrons
x10-14
cm
Why deuteration of a protein sample?
Deuteration largely avoids large incoherent scattering of hydrogen which contributes to the background of the diffraction images and enhances the visibility of hydrogen and water positions in the resulting neutron scattering density maps.
Hydrogenated and fully deuterated proteins
H σincoh = 80.27 barns D σincoh = 2.05 barns
H to D; incoherent scattering reduced and S/N ratio increased.
A-DNA/H2O A-DNA/D2O
H-atoms appear as negative peaks in high-resolution neutron Fourier maps, however, at medium resolution, cancellation can occur
(Courtesy of T. Forsyth, ILL)
Approx. 50% of a protein structure are hydrogen atoms.
With D-atoms, no cancellation of density but rather enhancement of positive nuclear density.
Incoherent scattering adds to background on detector, therefore reduces the S/N ratio of the data.
Fully deuterated proteins;
Smaller crystal volumes needed (~0.1mm3)
• Can address larger unit-cell problems• Aids the success of cryo-cooling the
crystal• Decreases the spatial overlap
problem of reflections
Exchanging all H for D aids the refinement and interpretation of the structure
The ILL-EMBL DeuterationLaboratory
Biopolymer synthesis
Crystallogenesis
Fermentation
D, 15N, 13C labelling of macromolecules
for neutron scattering and NMR
Photobioreactors
Dedicated P2 facilities
Molecular biology, cloning,
expression, purification
Proteomics
Neutron Protein Crystallography
Solution Scattering (SANS)
Fibre Diffraction
Inelastic Neutron ScatteringObservation of conformational
changes of single sub-unitswithin a complex
Localization of catalytic protons in enzymes Localization of water molecules in DNA
Studies of macromolecular dynamics
Deuterium labelling for neutron scattering experiments
LADI
D11D22
D19
IN
Neutron reflectivityStructure of model membranes,
and the interaction with peptides,proteins and DNA
D17
The ILL-EMBL Deuteration Facility
web-site: http://www.ill.fr/deuteration [email protected]
Why to improve the size and quality of crystals?
Large crystals are required to compensate for the weak flux of available neutron beams.
Neutrons
mm3
X-rays diffracted
X-rays
μm3
Neutrons diffracted
X-ray source (ESRF) much more (109x) intense than
neutron source (ILL)
( )2cell
sample2
0
VV.F.I
I ≈
DiffractionIntensity in Bragg reflections
Incident neutronintensity
Unit cellvolume
Structurefactor
Illuminated volume of crystal
Measured signal is directly proportional to the crystal volume
A methodology and an instrument for the temperature-controlled optimization of
crystal growth
allowing for control of the kinetics of the crystallization process by taking advantage of thermodynamics and generic features of the phase diagram.
Budayova-Spano et al., Acta Cryst. D63, 2007, 339-347
• Solubility measurement f(T)
• Identifying the favourable zone for ordered crystal growth (metastable zone) thanks to a change in temperature.
• Promotion of crystal growth by keeping the crystallization solution metastable during the process of crystal growth thanks to a change in temperature.
Rational physico-chemical approach based on knowledge of the phase diagram
A methodology for the temperature-controlled optimization of crystal growth
Budayova-Spano et al., Acta Cryst. D63, 2007, 339-347
Solubility measurements f(T)
Time
Solution proteinconcentration
by crystallization
by dissolution
• Protein solubility is obtained by following the concentration variation of super and under-saturated solutions seeded with small protein crystallites.
• This is done by removing aliquots and measuring the absorbance at 280nm.
• The protein concentration in equilibrium crystal/solution is measured and corresponds to the solubility at a given temperature.
Pro
tein
con
cent
ratio
n (m
g/m
l)
Temperature (ºC)
0
2
4
6
8
10
12
14
0 5 10 15 20 25 30
Cs H2O (T) = Cs D2O (T+7.2°C)
0
2
4
6
8
10
12
14
0 5 10 15 20 25 30Temperature (ºC)
Pro
tein
con
cent
ratio
n (m
g/m
l) Cs H2O (T)Cs D2O (T)
Cs H2O (T) > Cs D2O (T)
5% PEG 8000, 100mM NaCl, Tris-HCl 50mM, pH (pD) 8.5
Budayova-Spano et al., Acta Cryst. D63, 2007, 339-347Urate oxidase (Uox), involved in catalysis of the oxidation of uric acid to allantoin
Crystal growth of the seeds is maintained inside the metastable zone as long as possible thanks to the temperature variations as soon as the equilibrium crystal/solution is reached.
Improved Large Crystal Growth at 20 °C200μl: 5% PEG 8000, 100mM NaCl, Tris-HCl
50mM, init. prot. conc. 8mg/ml, pD 8.5
Total time 2 days, 1 image per 2 hours
0
2
4
6
8
10
0 5 10 15 20 25 30
Temperature (°C)
Prot
ein
c onc
entr
atio
n (m
g/m
l)
Direct Solubility
Improved Large Crystal Growth
U N D E R S A T U R A T I O N
SOLUBILITYCURVE= SATURATION
START
Metastablezone
END
Nucleation zone
S U P E R S A T U R A T I O N
Promotion of crystal growth by keeping the crystallization solution metastable: Urate oxidase (Uox), involved in catalysis of the oxidation
of uric acid to allantoin (Budayova-Spano et al., Acta Cryst. D63, 2007, 339-347)
Growing large crystals for neutronsCase of recombinant Uox complexed with a purine-type inhibitor (8-AZA)
• Neutron scattering density map (2Fo-Fc at 1.5 sigma) superposed with the current model of Uox-8-AZA• Clear density for D atoms and orientations of D2O molecules
Vfinal=1.8mm3
2.1Å resolution on LADI-ILL
Illustrating the high quality of the neutron Laue diffraction data collected from crystals grown via knowledge of the phase diagram.
Budayova-Spano et al., 2006 Acta Cryst. F62, 306-309.
0.5mm
Growing large crystals for neutronsCase of perdeuterated yeast inorganic pyrophosphatase, model system for
studying phosphoryl transfer reactions catalysed by multiple metal ions
1. Temperature variationto low temperature values(20°C => 5 °C) allows tostabilise and grow thecrystalline form of ourinterest
Start
1 month later
2 months later:≈ 0.15mm3
2 months later:≈ 0.7mm3
2. Crystal quality grown bythis method appears to bebetter than that of the seed(centre of the crystal)
200μl: 15% MPD, 1mM MnCl2, 1mM Pi, 30mM MES pD 6.0, prot. conc. 20mg/ml
Diffraction to 3Å resolutionon LADI-ILL
X-ray diffraction to 1.9Å
Budayova-Spano et al., ActaCryst. D63, 2007, 339-347
Growing larger better diffracting crystals for X-rays
Seeding at 35ºC
Crystallization Batch: 100μlProt. Quantity:33μl (800 μg)
Growth at 35ºC t=3days
Crystal cluster grown by hanging-drop vapour-diffusion technique at 20 ºCprot. conc. 24mg/ml
Diffraction to 2.5Å resolutionat id29 ESRF Growth at 30ºC
t=6 days
Diffraction to 1. 5Å resolutionat id29 ESRF
50μm (a) 50μm (b)
50μm (c) 50μm (d)
1.15M Na citrate, 100mM Tris-DCl pD 7.5
Transforming the clusters of crystals to the single crystals suitable for X-ray analysis
Case of perdeuterated human carbonic anhydrase II involved in catalysis of the hydration of carbon dioxide
Budayova-Spano et al., 2006 Acta Cryst. F62, 4-9.
• Investigating the phase diagram, controlling the nucleation and crystal growth of biomacromolecules, manipulating the solubility of seeded H/D – labelled crystals as a f(T)
• Regulating the temperature of the crystallization solution using control parameters determined in situ during the growth process (Novel multi-well crystal growth apparatus)
• Allowing for in situ observation by optical microscopy and sequential image acquisition, processing and storage
• Facilitating the convenient extraction of the protein crystals after growth, without causing any mechanical damage to them => using MICROMANIPULATOR
An instrument for the temperature-controlled optimization of crystal growth Budayova-Spano et al., Acta Cryst. D63, 2007, 339-347
Acknowledgements and partners• Stephen Cusack (EMBL Grenoble)• Peter Timmins (ILL Grenoble)
• François Dauvergne (EMBL Grenoble) Mechanics• Michel Audiffren & Thirou Bactivelane Electronics and
(CINaM CNRS Marseille) Software
• Marie-Thérèse Dauvergne (EMBL Grenoble) Production of perdeuteratedmaterial
• Françoise Bonneté (CINaM CNRS Marseille) Uox neutron• Bertrand Castro & Mohamed El Hajji diffraction project
(Sanofi-Aventis Montpellier)
• Adrian Goldman & Esko Oksanen Ppase neutron(University Helsinki, Finland) diffraction project
• Matthew Blakeley (ILL Grenoble) Data collection on LADI (ILL)
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