European
Molecular
Biology
Laboratory
Grenoble Outstation
Protein structure-function studies by
neutron crystallography
Monika Budayova-Spano
UJF – EMBL / ILL
[email protected]
Protons in biology
Knowing exactly where protons are, and how they are transferred
between macromolecules, solvent molecules and substrates is
crucially important for understanding many biological processes.
•Enzyme mechanisms; location of the
protons and hence protonation states of
key amino-acid residues at the active site
aids our understanding of catalytic
activity.
•Solvent structure; can play an
important role in the function of many
macromolecules. Direct determination of
the positions and orientations of bound
water molecules as D2O. Gain vital
information on hydrogen bonding
Trypsin
Vitamin
B12
Endothiapepsin
•H/D exchange; identification of solvent
accessible areas (dynamics)
•Ligand-binding interactions;
identification of key hydrogen bonds and
ligand protonation state.
1:1 co-crystal of BPY and thiodiglycolic acid
Neutron diffraction
X-rays:
Neutrons:
•Scattered by the electrons
•Scattered by the atomic nuclei
•Scattering factors proportional to the
number of electrons
•Scattering lengths depend on the nuclear forces and can be different for
isotopes of the same element e.g. Hydrogen and Deuterium
•Hydrogen and Deuterium hence scatter
X-rays very weakly
•Considerably less variation across the periodic table for neutron
scattering lengths, with H and D on similar scale to C, N, O and S.
•X-ray data to ~1.2 Å resolution required
– and even then the visibility can be
hampered by their degree of thermal
motion (B-factors > 10 Å2)
•Neutron diffraction data at medium resolution (1.5 – 2.5Å) can give
detailed information on the positions of the hydrogen and deuterium
atoms within a macromolecule and its bound solvent.
Isotope
•Negative values are
associated with a resonance
level that produces an extra
180º phase shift.
•Positive values of the
neutron scattering length, b,
reflect a 180º change in phase
between the incident and the
scattered neutron waves.
Atomic
Number
Neutron coherent
scattering length, b
(10-12 cm)
Neutron
incoherent
cross
section (Barns)
X-ray scattering factors
(10-12 cm)
sin θ = 0
1H
2H
1
(D)
sin θ / λ = 0.5 Å-1
-0.374
80.27
0.28
0.02
0.667
2.05
0.28
0.02
12
C
6
0.665
0.00
1.69
0.48
14
N
7
0.937
0.49
1.97
0.53
O
8
0.580
0.00
2.25
0.62
Mg
12
0.549
0.00
3.38
1.35
S
16
0.280
0.00
4.50
1.90
K
19
0.379
0.25
5.30
2.20
Mn
25
-0.375
0.40
7.00
3.10
26
1.012
0.00
7.30
3.30
16
24
32
39
55
56
Fe
Macromolecular neutron diffraction
•Total neutron scattering cross-section is the sum of two terms, the coherent and incoherent crosssections.
•Coherent cross-section gives rise to interference, while the incoherent cross-section serves only to
increase the background.
Large difference in the incoherent crosssection among isotopes
•Hydrogen has a very large incoherent
scattering cross-section of 80.27 Barns.
•As H-atoms make up ~50% of the atoms, a
large background signal is observed which
reduces the S/N ratio of the data and hence
the resolution limit.
• H-atoms give negative peaks in highresolution neutron Fourier maps, however, at
medium resolution, cancellation can occur
(C-H, N-H, H-O-H)
A-DNA/H2O
A-DNA/D2O
• Partial: Protiated crystals can be soaked in D2O or
grown in D2O – only exchanges those protons
attached to O or N
• Full: Perdeuterated crystals can be produced by
expression of the macromolecule from bacteria grown
on deuterated media – exchanges all H for D
•Deuterium has a incoherent cross-section
of 2.05 Barns, ~40 times less than Hydrogen.
•Replacing H with D dramatically reduces the
background and increases the S/N ratio and
hence the resolution limit.
• With D-atoms, all positive nuclear density,
no cancellation (C-D, N-D, D-O-D)
DEUTERATION
Early neutron diffraction studies
•Large crystal volumes of several mm3 were necessary
•Limited in asymmetric unit-cell volume (< 55000 Å3) that could be studied
•Monochromatic data collection took from 1 - 6 months for a single data set
•Only a few studies performed in ~15 years
Name
Lattice constants (Å) / Space group
Dmin (Å)
Crystal Volume,
Vxtal (mm3)
Unit-cell volume, Vuc
(Å3)
Asymmetric unit
volume, Va (Å3)
Vxtal/Va
Collection
Time (days)
Bovine Trypsin/MIP
54.9 x 58.6 x 67.5 / P212121
2.2
1.6
217,157
54289
295
-
Subtilisin
39.3 x 72.8 x 75.2 / P212121
2.0
2.7
215,150
53788
502
-
Perdeuterated met-Mb pH 6.2
(sperm whale)
64.5 x 30.9 x 34.8 β = 105.8° / P21
2.0
2.5
66,738
33369
749
-
2Zn Insulin
82.5 x 82.5 x 34.0 γ = 120°/ H3
2.2
3.0
200,413
22268
1347
-
Crambin
41.0 x 18.7 x 22.5 β = 90.8° / P21
1.5
1.4
17,249
8625
1623
-
met-Mb pH 5.6 (sperm whale)
64.6 x 31.0 x 34.9 β = 105.8° / P21
1.7
-
67,251
33626
-
-
Oxy-Mb pH 8.4 (sperm
whale)
64.5 x 31.1 x 34.9 β = 105.8°
/ P21
2.0
8.0/8.0
67,363
33682
2375
42
Ribonuclease A/uridine vanadate
30.3 x 38.4 x 53.7 β=106.4° / P21
2.0
18.9
59,939
29970
6306
-
BPTI (form II)
74.1 x 23.4 x 28.9 / P212121
1.8
8.0
50,111
12528
6386
140
MbCO pH 5.6 (sperm whale)
64.6 x 31.0 x 34.9 β = 105.8° / P21
1.8
24
67,251
33626
7137
-
HEW triclinic Lysozyme
27.3 x 32.0 x 34.3 α = 88.8°, β = 108.8°, γ =
111.6° / P1
1.4
20
26,212
26212
7630
~45
Ribonuclease A
30.2 x 38.4 x 53.3 β=105.9° /
P21
2.0
30.0
59,447
29724
10093
180
Vitamin B12 coenzyme at 15K
27.6 x 21.6 x 15.3 / P212121
0.9
5.0
9,121
2280
21930
35
Vitamin B12 coenzyme at 279K
27.8 x 21.7 x 15.4 / P212121
0.95
6.0
9,290
2323
25829
21
Key developments: Instrumentation - LADI-I
The Laue diffractometer, LADI-I
In the early Nineties, a neutron Laue diffractometer, LADI-I, was constructed at the ILL. The experimental
design of the instrument included two key differences compared to diffractometers previously used.
The neutron image plate: A large horizontal cylindrical area
detector made of neutron image plates (photostimulable
phosphor doped with Gadolinium [Gd2O3 doped
BaF(Br.I):Eu2+]) was designed that surrounds the sample
and allows many stimulated Bragg reflections to be recorded
simultaneously offering large coverage of reciprocal space.
Laue and Quasi-Laue Diffraction method: The sample is
illuminated by all available neutrons, maximising the flux at
the sample and stimulating large numbers of reflections
simultaneously over all incident wavelengths. Due to the
high background and problems of spatial overlap with a
white beam, a restricted wavelength band is extracted
from the white beam using Ni/Ti multilayer band pass
filters. Various Ni/Ti multilayer filters are available to select
the wavelength range (δλ/λ) and wavelength (λ) that is
best suited to the sample.
These combined improvements greatly
accelerated data collection rates making
smaller crystals and larger systems
accessible to neutron crystallography
Quasi-Laue diffraction
Scattering proportional to the λ2 so use longer λ neutrons i.e. cold neutrons
Laue – high flux but background very high
Quasi-Laue (using multilayer band-pass filter) - reduces background while
maintaining higher flux than monochromatic data collection
Tuning by displacement (2.8 Å < λcentre < 4 Å)
Other neutrons
Neutron (cold)
guide H142
Ni/Ti multilayer band
pass filter
1.
Full white
beam
2.
Selected
band-pass
using Ni/Ti
multilayer
filter
Selected wavelength
range of neutrons
The LADI-I instrument
LADI-I structures solved (1997-2007)
Name
Lattice constants (Å) / Space
group
Dmin
(Å)
Crystal Volume,
Vxtal (mm3)
Unit-cell volume, Vuc (Å3)
Asymmetric unit volume,
Va (Å3)
Vxtal/Va
Collection
Time
Perdeuterated Human
Aldose
Reductase/NADP+/IDD594 complex
50.1 x 67.1 x 47.9
β=92.4° / P21
2.2
0.15
160,885
80443
19
93
Urate oxidase/8-azaxanthin complex
81.3 x 96.3 x 105.6 / I222
2.1
1.8
826,762
103345
174
34.5
Xylose Isomerase
92.8 x 98.4 x 101.5 /
I222
2.2
4.0
926,849
115846
345
21
Endothiapepsin/H261 complex
43.1 x 75.7 x 42.9 β=97.0° / P21
2.1
3.5/3.0
138,926
69463
468
69
Trp repressor mutant (Val58 to Ile58)
53.6 x 53.3 x 32.7 / P21212
2.1
1.1
93,420
23355
471
13.5
Concanavalin A at 15K
89.2 x 86.1 x 61.6 /
I222
2.5
1.6/5.6
473,095
59137
609
34
HEW triclinic Lysozyme
27.3 x 32.0 x 34.3 α = 88.8, β =
108.8, γ = 111.6 / P1
1.7
2.0/2.0
26,211
26211
763
14
W3Y rubredoxin at 15K (Pf)
34.1 x 34.9 x 43.7 / P212121
1.7
1.4
52,007
13002
1077
7.5
HEW monoclinic Lysozyme
28.0 x 62.9 x 60.3 β=90.7° / P21
2.1
6.3
106,192
53096
1187
13
HEW tetragonal Lysozyme
79.1 x 79.1 x 36.6 / P43212
2.0
6.0
228,919
28615
2097
10
Concanavalin A
88.7 x 86.5 x 62.5 / I222
2.4
15.0
479,534
59942
2502
20
Concanavalin A
89.4 x 87.3 x 63.1 / I222
2.2
21.0
492,472
61559
3411
15
Coenzyme cob(II)alamin (B12r)
16.0 x 21.9 x 26.8 / P212121
1.43
8.2
9,391
2348
34923
1.5
Highlights from LADI-I structures
Aldose Reductase: first full deuterated enzyme structure from LADI-I and with
remarkably smaller crystal volume (0.15mm3) than previously used
Concanavalin A at 15K: first ever published cryo- neutron protein structure. Twice as
many D2O molecules visible at 15K compared to 293K (use of a He displex cryostat)
Key developments: Instrumentation - Diffractometer upgrade, LADI-III
•For LADI-I image plates and readout system were
located on the exterior of the drum
Sample holder
•For LADI-III image plates and readout system
located inside the drum so that the IP is read from
the ‘correct’ side.
•Drum diameter (400mm) and length (450mm) both
increased to aid in reduction of spatially
overlapped reflections and to reduce the overall
background scatter per pixel
Internal read/erase
system
Neutron image
Plates
Cylindrical
drum detector
Active area 1245 × 450 mm2
Angle subtended 172° in T, 49° in v
LADI-I versus LADI-III comparisons
Comparisons between tetragonal lysozyme data collected on LADI and LADI-III
(using the same crystal and aperture size).
Several comparisons;
LADI stats to 2.0 Å overall
and in parentheses for
highest resolution shell
LADI-III stats to 2.0 Å overall and in
parentheses for highest resolution
shell
12h exposure with 2.9mm aperture
3821 observations
I/σ(I) = 6.5
4501 observations
I/σ(I) = 10.5
12h exposure on LADI vs. 4h exposure
on LADI-III
3821 observations
I/σ(I) = 6.5
4365 observations
I/σ(I) = 8.0
Data collected in total time of 36h
R-merge = 14.3 (18.3)
% complete = 53.2 (34.3)
Multiplicity = 1.7 (1.5)
I/σ(I) = 4.7 (3.1)
R-merge = 16.1 (18.0)
% complete = 71.8 (51.2)
Multiplicity = 4.3 (2.6)
I/σ(I) = 7.4 (3.8)
Equivalent coverage of reciprocal
space (40°)
Time = 84h
% complete = 66.1 (49.0)
Multiplicity = 2.6 (1.9)
I/σ(I) = 5.7 (3.4)
Time = 40h
% complete = 72.7 (52.1)
Multiplicity = 4.7 (2.7)
I/σ(I) = 7.8 (3.9)
LADI-III enables us to collect data to higher resolution, in
shorter times and from larger unit cell systems!!!
LADI-III structures from June 2007 – July 2008
Name
Lattice constants (Å) / Space
group
Dmin (Å)
Crystal
Volume, Vxtal
(mm3)
Unit-cell volume,
Vuc (Å3)
Asymmetric unit
volume, Va (Å3)
Vxtal/Va
Collection Time
(days)
A-DNA d(ACCCCGGGGT)
32.8 x 32.8 x 78.3 /
P6122
2.3
0.06
72,954
12159
49
9.0
Cytochrome c peroxidase
51.6 x 76.7 x 107.2 / P212121
2.4
0.8
424,268
106067
75
5.6
Cex catalytic domain (glycosyl-enzyme
intermediate)
88.2 x 88.2 x 81.2 / P41212
2.5
0.7
631,674
78959
89
13.5
Urate oxidase/Urate complex
80.1 x 96.0 x 105.2 / I222
2.3
0.9
808,946
101119
89
18
Perdeuterated Type-III
Antifreeze protein
32.5 x 39.4 x 45.3 /
P212121
1.85
0.13
58,007
14502
90
18
Proteinase K
67.7 x 67.7 x 107.3 / P43212
2.4
0.8
491,787
61473
130
2.5
A Urate oxidase complex
80.1 x 96.0 x 105.2 / I222
2.2
1.8
808,946
101119
178
5.3
γ-Chymotrypsin
69.5 x 69.5 x 97.7 / P42212
2.0
1.3
471,915
58989
220
8.0
Thaumatin
57.8 x 57.8 x 149.6 / P41212
2.1
1.4
499,790
62474
224
10.5
Urate oxidase/8-azaxanthin complex
80.1 x 96.0 x 105.2 / I222
1.9
4.0
808,946
101119
395
6
D-Xylose isomerase/D-xylitol complex
92.9 x 98.9 x 101.8 / I222
2.0
8.0
935,319
116915
684
12
Perdeuterated Rubredoxin (Pf)
33.9 x 34.9 x 43.5 /
P212121
1.75
1.4
51,465
12866
1088
0.5
Perdeuterated Rubredoxin (Pf)
33.9 x 34.9 x 43.5 / P212121
1.65
4.0
51,465
12866
3109
5.5
Selectively CH3-protonated Perdeuterated
Rubredoxin (Pf)
33.9 x 34.9 x 43.6 / P212121
1.69
4.1
51,584
12896
3179
3.5
•In the past year neutron Laue data sets have been collected using LADI-III for several
new macromolecules.
•As many structures as were done in the previous 10 years!
•Due to detector improvements (LADI-III readout design) and advances in sample
preparation (perdeuteration and the rational growth of large protiated crystals).
Key developments: Sample preparation - Perdeuteration
• Perdeuteration allows the use of smaller crystal volumes, the ability
to collect data from larger systems, shorter data collection times and
potentially higher resolution data.
• Perdeuterated crystals aid the interpretation of the nuclear maps
and the smaller crystal volumes required reduce the number of
spatially overlapped reflections on a fixed-radius detector.
The ILL-EMBL Deuteration Laboratory
Molecular
biology, cloning,
expression,
purification
D, 15N, 13C labelling
of macromolecules
for neutron
scattering and NMR
Fermentation
Crystallogenesis
Dedicated P2
facilities
Biopolymer
synthesis
Photobioreactors
http://www.ill.fr/deuteration
Proteomics
Key developments: Sample preparation – Growth of large
crystals
Structure
factor
Required to compensate for the weak
flux of available neutron beams.
X-rays diffracted
μm3
X-rays
Neutrons diffracted
mm3
Diffraction
Intensity in
Bragg
reflections
2
I≈
I0 .F .Vsample
(Vcell )2
Incident
neutron
intensity
Illuminated
volume of
crystal
Unit cell
volume
Measured signal is directly
proportional to the crystal volume
Neutrons
As a consequence of the low neutron fluxes (some 106 – 107 times less intense
than the photon fluxes from X-ray synchrotron sources), it has been, until rather
recently, a necessity to have an extremely large crystal (>1mm3); the actual crystal
volume required depending on the sample unit-cell volume. Growth of
macromolecular single crystals with volumes of several mm3 can be difficult.
Key developments: Sample preparation – Growth of large
crystals
For crystallization
D2O ≠ H2O
deuterated protein ≠ protiated protein
Budayova-Spano et al., Acta Cryst. D63, 2007, 339-347
Protiated urate oxidase in ∼ 5% PEG 8000, 100mM NaCl, Tris-HCl 50mM, pH (pD) 8.5
14
14
Cs H2O (T)
Cs H2O (T) > Cs D2O (T)
10
8
6
4
2
0
0
5
10
Cs H2O (T) = Cs D2O (T+7.2°C)
12
Cs D2O (T)
Protein concentration (mg/ml)
Protein concentration (mg/ml)
12
15
Temperature (ºC)
20
25
30
10
8
6
4
2
0
0
5
10
15
Temperature (ºC)
20
25
30
Rationalizing Biomacromolecular Crystallization:
a physico-chemical approach
A methodology and an instrument
allowing for control of the kinetics of the crystallization
process by taking advantage of thermodynamics and
generic features of the phase diagram.
A methodology for the temperature-controlled
optimization of crystal growth
Budayova-Spano et al., Acta Cryst. D63, 2007, 339-347
Urate oxidase / 8aza
Improved Large Crystal Growth
Protein concentration (mg/ml)
10
200μl: 5% PEG 8000, 100mM NaCl, Tris-HCl
50mM, init. prot. conc. 8mg/ml, pD 8.5
START
Direct Solubility
Metastable
zone
8
Nucleation zone
6
SUPERSATURATION
4
2
SOLUBILITY
CURVE
= SATURATION
END
0
0
UNDERSATURATION
5
10
15
20
25
30
Temperature (°C)
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 °C
Total time 2 days, 1 image per 2 hours
An instrument for the temperature-controlled optimization of
Budayova-Spano et al., Acta Cryst. D63, 2007, 339-347
crystal growth
• 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
Urate oxidase complexes
•Urate oxidase (Uox) is an enzyme
which catalyses the oxidation of
uric acid to allantoin.
•Uox is used as a protein drug to
reduce toxic uric acid accumulation
and to resolve the hyperuricaemic
disorders occurring during
chemotherapy.
Details of the positions of the H
atoms within the active site, critical
for a complete understanding of the
mechanism are unknown.
Using a novel method and apparatus
that allows the manipulation of the
kinetics of the crystallization
process, large crystals (>> 2mm3) of
several Uox/inhibitor complexes have
been grown.
Large
(4mm3)
crystal of
Uox/8aza
Urate oxidase/8-azaxathin data collection
Overall data quality to 1.9 Å
•R-merge = 14.1 (23.6, 7.6)
35 images in 3 orientations using 4h
exposures with crystal of volume
4.0mm3 and φstep = 7°
•I/σ(I) = 9.2 (2.6, 18.4)
Total time = 5.8 days
•% complete = 73.2 (45.2, 97.1)
Wavelength range = 3.25 – 4.35 Å
(high resolution, low resolution)
•Multiplicity = 5.8 (3.0, 7.5)
Urate oxidase/8-azaxathin nuclear density
Urate oxidase has one of the largest primitive unit-cell volumes (space group
I222, unit-cell parameters a = 80.1, b = 96.0, c = 105.2 Å) and molecular weights
(135 kDa for the homotetramer) so far successfully studied with neutrons.
A second Urate oxidase complex
•Large crystals also grown of a
second Uox complex.
•Data collected on LADI-III from
crystal with volume
using 6h exposures.
1.8mm3
•21 images in 2 orientations using
φstep = 7°
•Total time = 5.3 days
Overall data quality to 2.2 Å
(high resolution, low resolution)
•R-merge = 11.7 (20.4, 5.6)
•I/σ(I) = 8.3 (1.8, 25.2)
•% complete = 70.6 (53.8, 90.9)
•Multiplicity = 3.1 (2.2, 3.5)
Room temperature X-ray data
collected on same crystal and
joint X+n refinement underway.
Key developments: Joint X-ray and neutron refinement
• By combining diffraction data from both X-ray and neutron
techniques, the data-to-parameter ratio is increased while the
influence of systematic errors can be reduced.
• Joint X+n refinement strategies thus make it possible to allow full
refinement of all the atoms within the structure leading to a better
description.
• Two programs, nCNS and PHENIX have been developed which allow
such joint refinement strategies.
Highlights from LADI-III structures thus far…
• Fast (<7days) data collection for several macromolecules
e.g. RdPf, Proteinase K, Urate Oxidase complexes,
Cytochrome c Peroxidase
• Medium-resolution data from small (<1mm3) crystal
volumes e.g. AFP (0.13mm3), A-DNA (0.17mm3), CcP
(0.8mm3), Cex (0.7mm3), Proteinase K (0.8mm3)
• Medium-resolution data from large unit-cell systems e.g.
Xylose Isomerase complex, Urate Oxidase complexes,
Thaumatin, γ-Chymotrypsin, Cex, Proteinase K
Acknowledgements
Matthew Blakeley (ILL)
Esko Oksanen (University of Helsinki)
François Dauvergne (EMBL)
Marie-Thérèse Dauvergne (EMBL)
Stephen Cusack (EMBL)
Peter Timmins (ILL)