irene margiolaki - brookhaven national laboratoryprotein crystallography via powder diffraction...
TRANSCRIPT
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Powder Diffraction on proteins
Irene Margiolaki
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ESRF
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PresentationA Challenging Project
Protein Crystallography via powder diffraction
Developments
• Improvements in data quality
• Novel methods for data analysis: Case studies of small proteins
• Cryocooling of protein powder samples
Perspectives
Projects & Collaboration with Industry
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Use the entire powder
pattern on a point by
point basis
Bragg
Reflections
Background
Peak shapes
Peak positionsPeak Intensities
Cell parameters
Structure
Positions
Occupancies
Thermal factors
Microstructure
Size, Shape, Strain
Composition
(“Phase” analysis)
Non-crystalline
components
The peak overlap
problem makes all of
this more difficult!
Rietveld Methods….
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Protein CrystallographyTRADITIONAL METHOD:
Single Crystal Diffraction
•Gives 3D information about crystal
structure
•Very small amount of protein
required
HOWEVER
•Can be very difficult to grow large
enough single crystals of some
proteins
•Very specific crystallisation
conditions may not represent the
natural environment
COMPLEMENTARY METHOD:
Powder Diffraction
•Three dimensional information is
collapsed down into 1D – loss of
information!
•Larger protein sample required
HOWEVER
•Polycrystalline powder can often
be obtained when a good single
crystal cannot
•More crystallisation conditions
possible
•Phase mixtures and phase
transitions can be observed in-situ
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Proteins Characterised successfully using Powder Data
41 structural models deposited in the Protein Data Bank (PDB)
employing powder diffraction
http://www.rcsb.org/pdb/home/home.do
15 proteins characterised using synchrotron data
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Improvements in data quality
Exploring different Instruments
The use of multiple data sets
Combining different detectors
at the same time
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ID31: High Resolution Powder
diffraction Beamline
Detection of scattered beam using 9
APD detectors and 9 Si(111) analyser
crystals
Thanks to J.-L. Hodeau, M. Anne, P. Bordet, A. Prat, CNRS, Grenoble.
Parallel beam geometry
Experimental Conditions:
He- Cryostat for measurements down to 3 Κ.
“Hot-air blower”: Measurements up to 950 C
Oxford Cryosystems Cryostream (Cold Nitrogen
Gas blower) Measurements in the temperature range:
80 - 500 K
“Mirror Furnace ”: measurements up to ~1500ºC (Pt
capillaries)
Translation stages (XYZ) for in situ measurements of
residual stress.
A. N. Fitch, J. Res. Matl. Inst. Stand. Technol.
109, 133-142 (2004)
Na2Ca3Al2F14
= 0.60101 Å
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ID31 data - Pawley in TOPAS
HEWL, λ= 1.251209(6)Å
P43212: a= 79.2688(3) Å, c= 37.9573(2) Å
2956 Extracted Intensities up to 3Å resolution.
Margiolaki & Wright, Acta Cryst. A64, 169-180 (2008)
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Area detectors available at ID11/ESRF
•Materials Science Beamline
•High energy = small angles
•Low absorption
•Aluminium compound refractive lenses
– Focus at detector
– Transfocator: Snigirev/Rossat
•Variety of area detectors
– Bruker smart 6500
– ESRF Frelon
– Trixel / Pixium
Focus to ~10μm at detector
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ID11 using a FReLon camera
Exploring different detection systems
ID31
ID11
ID11
SNBL-BM01A
zoom
HEWL data collected at the analyzer crystal beam line- ID31 (red line), the area detector beam line-
ID11 using a CCD camera (green line). Inset: Magnification of the low angle data,
The area detector station of SNBL- BM01A.
I. Margiolaki, J. P. Wright, A. N. Fitch, G. C. Fox, A.
Labrador, R. B. Von Dreele, K. Miura, F. Gozzo, M.
Schiltz, C. Besnard, F. Camus, P. Pattison, D.
Beckers, T. Degen,
Z. Kristallogr. Suppl. 26 (2007) 1-13
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The multi-data-set Rietveld refinement for HEWL samples at pH 6.23, 5.73, 4.80, 3.83, crystallised
at 277 K.
Basso, S., Fitch, A. N., Fox, G. C., Margiolaki, I. & Wright, J. P. (2005). Acta Cryst. D61, 1612–1625.
Different pH conditions
Multi data set refinement
p H
3 .5 4 .0 4 .5 5 .0 5 .5 6 .0 6 .5
Te
tra
go
na
l
La
ttic
e P
ara
me
ters
(Å
)
3 7 .8
3 7 .9
3 8 .0
3 8 .1
3 8 .2
7 8 .6
7 8 .8
7 9 .0
7 9 .2
7 9 .4
Vo
lum
e/
mo
lec
ule
in t
he
un
it c
ell
(Å
3)
2 9 6 0 0
2 9 6 5 0
2 9 7 0 0
2 9 7 5 0
2 9 8 0 0
2 9 8 5 0
a
c
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• Molecular Replacement
• Isomorphous Replacement
Traditional Methods in protein crystallography
• CCP4, http://www.ccp4.ac.uk/
• CCP14, http://www.ccp14.ac.uk/
Combination of Software designed for single crystal and powder
diffraction data
• PRODD, Wright et al. Z. Kristallogr. Suppl. 26 (2007) 27-32
• GSAS, Von Dreele, R. B. (2007). J. Appl. Cryst. 40, 133–143 & Basso et al., Acta Cryst.
D61, 1612-1625 (2005)
Improved methods for Intensity extraction and refinement via
the use of multiple profiles
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After purification the SH3.2 domain spontaneously
formed a microcrystalline material suitable only
for powder diffraction measurements
Second SH3 domain of Ponsin: SH3.2
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Data collection
• Two samples A & B
• Spinning glass capillaries
d=1.5 mm
• Mounted on the axis of
the diffractometer
• Patterns were measured
with a period of 2.0 min
• Beam size: 1.5 mm2
(1.5 mm 1.0 mm)
• Photon flux on sample
~ 3 1012 photons s-1 mm-2
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Procedure followed for data analysis for SH3.2
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•Anisotropic peak shifts with radiation
damage
•Classification of scans using cluster analysis (Pycluster)
•Multi-pattern fit (prodd)
•Use of likelihood function to equi-
partition intensities
P212121, a = 24.80, b = 36.38, c = 72.25 Å
Intensity Extraction for SH3.2 domain of ponsin
J. P. Wright, A. Markvardsen and I. Margiolaki
Z. Kristallogr. Suppl. 26 (2007) 27-32
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Completeness & correlations to SX
Quality of extracted intensities
Combined fit
Single patterns
Very similar to completeness
3Å3Å
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Molecular replacement
--- Summary ---
S_ RF TF theta phi chi tx ty tz TFcnt Rfac Scor
S___1__1 1 58.07 144.99 102.00 0.161 0.266 0.181 3.47 0.527 0.377
S___6_15 2 145.90 144.44 92.50 0.361 0.166 0.366 5.14 0.595 0.238
S___2_10 3 125.77 -81.54 96.49 0.212 0.061 0.165 2.29 0.627 0.192
S___4__7 4 51.52 58.03 125.28 0.456 0.267 0.351 3.25 0.612 0.191
S___7__5 5 132.09 -179.53 72.05 0.167 0.366 0.214 1.40 0.621 0.184
S___8_10 6 175.99 -179.51 84.91 0.406 0.101 0.391 2.09 0.615 0.178
S__10_13 7 71.94 -151.69 19.36 0.378 0.216 0.318 1.78 0.625 0.177
S___5__5 8 62.17 -139.62 106.19 0.279 0.485 0.198 2.21 0.624 0.171
S___9_11 9 80.99 -179.11 79.75 0.284 0.452 0.132 1.62 0.616 0.165
S___3_13 10 30.37 76.91 178.57 0.346 0.253 0.137 2.17 0.627 0.146
Two models were tested
Solution always obvious
Solution Score for two different models
Molrep results:
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Initial electron density maps
2Fo-1Fc (blue at 1σ) and 1Fo-Fc (red at -2.5σ and green at 2.5σ) electron density maps, as determined directly after the
molecular replacement. The residues represented in grey stick carbon atoms correspond to the molecular replacement
model used for the calculation of the maps, while the residues in green color carbon atom sticks represent the final refined
model.
Initial model from pdb Final model after rebuilding and refinement
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λ= 0.8012034(76) Å, exposure time: 2 min.
Sample A: a=24.70420(9) Å
b= 36.42638(14) Å
c= 72.09804(26) Å
λ= 1.252481(32) Å, exposure time: 2 min.
ID31 Data
λ= 1.252481(32) Å, exposure time: 4 min.
Sample B: a= 24.79017(7) Å
b= 36.35407(12) Å
c= 72.22940(32) Å
λ= 1.251209(40) Å, exposure time: 2 min.
4 dataset restrained refinement
GSAS: Larson, A. C., Von Dreele, R. B. “General Structure Analysis System (GSAS) 2004” (Los Alamos National Laboratory Report LAUR).
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Selected regions of the final refined structural model in stick representation
and the corresponding total omit map contoured at 1σ.
544 protein atoms and 36 water molecules were identified
in total OMIT and difference electron density maps.
The Second SH3 domain of Ponsin
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Powder-diffraction structure of the ponsin SH3.2 domain. (A) Ribbon representation of the SH3.2 indicating the secondary structure elements of the domain. The main hydrophobic residues of the
binding interface as well as the positions of the n-Src and RT loops are indicated. (B) Electrostatic potential representation of domain identifying additionally the water molecules as red spheres.
SH3.2: The final model
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Structure ValidationERRAT:
http://nihserver.mbi.ucla.edu/ERRATv2/
+----------<<< P R O C H E C K S U M M A R Y >>>----------+
| |
| final2 2.8 67 residues |
| |
| Ramachandran plot: 98.2% core 1.8% allow .0% gener .0% disall |
| |
| Gly & Pro Ramach: 0 labelled residues (out of 10) |
| Chi1-chi2 plots: 0 labelled residues (out of 43) |
| |
| Main-chain params: 6 better 0 inside 0 worse |
| Side-chain params: 5 better 0 inside 0 worse |
| |
| Residue properties: Max.deviation: 2.7 Bad contacts: 0 |
+| Bond len/angle: 4.8 Morris et al class: 1 1 3 |
| |
| G-factors Dihedrals: -.24 Covalent: -.17 Overall: -.18 |
| |
| M/c bond lengths:100.0% within limits .0% highlighted |
| M/c bond angles: 80.1% within limits 19.9% highlighted |
+| Planar groups: 95.5% within limits 4.5% highlighted |
| |
+----------------------------------------------------------------------------+
+ May be worth investigating further. * Worth investigating further.
PROCHECK:http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.htm
l
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Comparison with the single crystal model
I. Margiolaki, J. P. Wright, M. Wilmanns, A. N. Fitch & N. Pinotsis.
JACS 129 (38): 11865-11871 SEP 26 2007
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Test systems
protein
Hen
egg-white
Lysozyme
(HEWL)
Pancreatic
porcine
Elastase
(PPE)
Molecular weight
Unit-cell (Å)
Space-group
14.4 kDa
a=b=79.2 c=38.0
P43212
24.8 kDa
a=51.8 b=57.9 c=75.3
P212121
Relative root
mean square
intensity change
(Crick and
Magdoff)
Gd (Z=64) : 0.40 U(Z=92) : 0.43
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Low resolution phasing in Gd derivative of lysozyme
• Sites from shelxd
• Refined in sharp
• Followed by density
modification
• Figure shows solvent
mask from density
modification
METHOD: Single Isomorphous Replacement (SIR)
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Gd-HEWL
Solvent channel found from map
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Heavy atom detection
Elastase
Lysozyme
Isomorphous
Patterson map
Direct methods
(SHELXD)Harker sections in
the Patterson map
for elastase.
C. Besnard et al.
Z. Kristallogr. Suppl. 26 (2007) 39-44
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Computed maps for Elastase-U
J. P. Wright et al. J. Appl. Cryst. (2008)Margiolaki, I. & Wright, J. P., Acta Cryst. (2008). A64, 169–180
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The quality of the density maps
• The correlations with the calculatedmaps are better for PPE than for HEWL.
0.28010.13944.09
-0.01760.20374.094.2
-0.06870.24154.24.31
0.08880.25734.314.44
-0.12040.23824.444.58
-0.23240.23734.584.74
0.05080.32374.744.93
-0.05730.29944.935.16
0.01280.34325.165.43
0.20460.44325.435.77
0.23170.30325.776.21
0.42360.40096.216.84
0.23550.45946.847.82
0.31280.33887.829.85
0.45550.54599.85inf
0.28010.13944.09
-0.01760.20374.094.2
-0.06870.24154.24.31
0.08880.25734.314.44
-0.12040.23824.444.58
-0.23240.23734.584.74
0.05080.32374.744.93
-0.05730.29944.935.16
0.01280.34325.165.43
0.20460.44325.435.77
0.23170.30325.776.21
0.42360.40096.216.84
0.23550.45946.847.82
0.31280.33887.829.85
0.45550.54599.85inf
PPE HEWLResolution (Å)
SIR
0.0489
0.2504
0.0355
0.0985
0.2409
0.0076
0.1894
0.0597
0.1325
0.4848
0.4164
0.6045
0.4796
0.7103
0.7686
HEWL
MIR
YES
SIR
0.0489
0.2504
0.0355
0.0985
0.2409
0.0076
0.1894
0.0597
0.1325
0.4848
0.4164
0.6045
0.4796
0.7103
0.7686
HEWL
MIRSIR
0.0489
0.2504
0.0355
0.0985
0.2409
0.0076
0.1894
0.0597
0.1325
0.4848
0.4164
0.6045
0.4796
0.7103
0.7686
HEWL
MIR
0.0489
0.2504
0.0355
0.0985
0.2409
0.0076
0.1894
0.0597
0.1325
0.4848
0.4164
0.6045
0.4796
0.7103
0.7686
HEWL
MIR
YES
• Can we improve them by using more derivatives?
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The HEWL MIR density map
Images created by Sebastian Basso
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The secondary structure
• Using FFFEAR we are able to locate all the
four helices of the lyzosyme structure out of
the five best fitted helices found by the
program using the standard fragment library.
Basso et al. In Preparation
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Author - Title (Footer) 34
Effect of radiation damage at Room temperature
0-60sec
1-2 min
2-3 min
3-4 min
4-5 min
5-6 min
6-7 min7-8 min
8-9 min
9-10 min
Courtesy: Yves Watier
10 mm
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Author - Title (Footer) 35
Lattice parameters shift during data collection
Lattice parameters shift during collection
Effect of radiation damage at Room temperature
Courtesy: Yves Watier
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Author - Title (Footer) 36
• D-spacing useful is larger.
• No ice peaks.
• Longer collection is possible.
• No radiation damage effects on lattice parameters while cooled.
• Just one position in the capillaries is shot, reducing greatly the
amount of sample needed.
Cryocooling- Goals
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Cryocooling
• Vast majority of single
crystal structures
collected at low
temperature
• Radiation damage
reduced
• Small reduction of B-
factors
Translating sample spinner at ID31 (Fitch/Rossat)
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Y. Watier et al.
Acta Cryst. (2008). A64, C312
Successful cryocooling of Insulin
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Industry
• Novo Nordisk – Copenhagen
(Human Insulin)
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Cubic
Rhombohedral R6
Rhombohedral T3R3f
Monoclinic
Tetragonal
Tetragonal
Rhombohedral T6
Cubic
Rhombohedral R6
Rhombohedral T3R3f
Monoclinic
Tetragonal
Tetragonal
Rhombohedral T6
Cubic
Rhombohedral R6
Rhombohedral T3R3f
Monoclinic
Tetragonal
Tetragonal
Rhombohedral T6
• New insulin crystal forms identified
Principal Component Analysis (PCA)
~80 years after the biological function of insulin was discovered
• Norrman, M. et al., (2006) J. Appl. Cryst 39 391-400
• Margiolaki, I. & Wright, J. P., Acta Cryst. (2008). A64, 169–180
• Knight, L. et al. (In Preparation)
New
New
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T6 Human Insulin
2Th Degrees232221201918171615141312111098765432
Inte
nsity (
a.u
.)
3 000
2 800
2 600
2 400
2 200
2 000
1 800
1 600
1 400
1 200
1 000
800
600
400
200
0
-200
-400
-600
hkl_Phase 0.00 %
2Th Degrees23222120191817161514131211
Inte
nsity (
a.u
.)
600
580
560540
520
500
480460
440
420
400380
360
340
320300
280
260
240220
200
180160
140
120
10080
60
40
200
-20
-40
-60
hkl_Phase 0.00 %
ID31- λ= 1.24965(2) Å
R3, a= 81.7531(4) Å, c= 34.0688(3) Å
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pH
5.75 7.0 7.05 7.707.356.0
P212121
C2221
C2 P21
R3
Phase transitions as a function of pH
C2/P21
a=112 Å
b=81.5 Å
c=336 Å
a=59 Å
b=220 Å
c=224 Å
a=100 Å
b=61 Å
c=63 Å
• Norrman, M. et al., (2006) J. Appl. Cryst 39 391-400
• Margiolaki, I. & Wright, J. P., Acta Cryst. A (In Press)
• Knight, L. et al. (In Preparation)
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Industry
• Sanofi Aventis – Paris
(Urate Oxidase)
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212019181716151413121110987654321
19,000
18,000
17,000
16,000
15,000
14,000
13,000
12,000
11,000
10,000
9,000
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
-1,000
-2,000
-3,000
-4,000
hkl_Phase 0.00 %
hkl_Phase 0.00 %
22212019181716151413121110987654
2,700
2,600
2,500
2,400
2,300
2,200
2,100
2,000
1,900
1,800
1,700
1,600
1,500
1,400
1,300
1,200
1,100
1,000
900
800
700
600
500
400
300
200
100
0
-100
-200
hkl_Phase 0.00 %
hkl_Phase 0.00 %
2Th Degrees65.85.65.45.254.84.64.44.243.83.63.43.232.82.6
Inte
nsity (
Co
un
ts)
5,000
4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
0
-500
hkl_Phase 0.00 %
ID31- λ= 1.300870(15) Å, LeBail fit in TOPAS
I222, a= 80.1888(5) Å, b= 96.2721(5) Å, c= 105.5448(5) Å
ID31 Powder Data
24/11/07Uox-AZA, 0% PEG, pH 7.5
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87,576,565,554,543,532,521,51
2 500
2 000
1 500
1 000
500
0
-500
hkl_Phase 0.00 %
2θ
inte
nsity
Space group P21212
V =1437603.9674 70.2393ų
a = 133.7876 ± 0.00397Å
b = 136.0288 ± 0.00366Å
c = 78.9937 ± 0.00221Å
New phase of urate oxidase
S. Dagogo et al. Acta Cryst. (2008). A64, C200
I. Collings et al. In Preparation
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Current Projects
• Characterisation of protein structures for antiviral drug
design, AFMB Marseille.
(Replication proteins from emerging viruses: Chikungunya, SARS, H1N1…)
• Studies of proteins from insect viruses/ virions from the
Baculoviridae family, Univ. of Auckland. (CPV’s)
• Amyloid- like synthetic peptide analogues of silkmoth
chorion proteins, Univ. of Athens.
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Acknowledgments
EMBL - Hamburg
Matthias Wilmanns
Nikos Pinotsis
ESRF
Andy Fitch (ID31)
Jon Wright (ID11)
Yves Watier (ID31)
Sotonye Dagogo (ID31)
Sebastian Basso (ID31)
Mark Jenner (ID31)
Lisa Knight (ID31)
Gavin Fox (BM16)
The ID31 team
CCP4 & CCP14
APS
Bob Von Dreele
IBS - Grenoble
Richard Kahn
EPFL
Marc Schiltz
Celine Besnard
Univ. of Manchester
John Helliwell
Novo Nordisk
Gerd Schluckebier
Mathias Norrman
CRMCN, Marseille
Françoise Bonneté
Marion Giffard
IUCr
Yuji Ohashi
Henk Schenk
Sanofi - Aventis
Bertrand Castro
M. El Hajji
PΑNalytical
BRUKER
SPring8
Keiko Miura
AFMB- Marseille
Nicolas Papageorgiou
Bruno Canard