WIR SCHAFFEN WISSEN — HEUTE FÜR MORGEN
Dr. Tim Grüne :: Paul Scherrer Institut :: [email protected]
Structural Chemistry and Structural Biology with Electrons
Anorganisch–Chemisches KolloquiumGeorg–August–Universität Göttingen12th December 2016
Tim Grüne IAC Kolloquium Göttingen
1 - Motivation
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The Novartis Library
• 2,000,000 compounds of potential drug targets
• 30-40% suitable for X–ray powder analysis
• 10% suitable for single crystal X–ray analysis
Dr. Trixie Wagner (2012)
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Single Organic Crystals from Powder
Novartis IRELOH:
Ø= 1,700nm = 1.7µm
Novartis EPICZA:
Ø= 500nm = 0.5µm
Novartis EPICZA:
Ø= 650nm = 0.65µm
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Zeolite Crystals at their Working Size
≈ 300nm Si-only zeolite type MFI (Pnma, 20.1Å, 19.7Å, 13.1Å)
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Crystalline Disorder — a Matter of Size?
K. Dalle, T Gruene, S Dechert, S Demeshko, and F Meyer, A weakly coupled biologically relevant CuII2 (µ −η1 : η1 −O2) cis-peroxo adduct
that binds side-on to additional metal ions JACS (2014), 136, 462–46
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2 - Electrons as Radiation Source
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X–ray Interaction with Matter
Interaction of X–rays at 12keV with 100µm
soft tisseA Transmission 96.6%B Photoabsorption 3.0%C Elastic Scattering 0.2%D Compton Scattering 0.2%
Red: Radiation damage Green: Diffraction
Every diffracting phtoton is accompanied by
16 damaging photons
Illustrative summary of x-ray and Îs-ray interactions. JA Seibert & JM Boone, J.Nucl. Med. Technol. 2005;33:3-18
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Electron Properties
1. wave–particle dualism cf de Broglie wavelength
2. typical electron energy: 100–300keV (200keV = 0.02508Å)
3. suitable for, but also require small samples: 100keV: sample < 100nm, 200keV: thickness < 300−500nm
4. electrons interact with charge: map = electrostatic potential inside crystal
5. strong interaction: multiple scattering events do occur
6. require high vacuum throughout (10−8mbar)
7. no anomalous signal (?)
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Electron Scattering
0
1
2
3
4
5
6
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
5.0 2.5 1.67 1.25 0.83 0.625
e- sca
tterin
g
sin(θ)/λ = 1/(2d) [1/Å]
HCNOAlSi
0
2
4
6
8
10
12
14
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
5.0 2.5 1.67 1.25 0.83 0.625
X-r
ay s
catte
ring
sin(θ)/λ = 1/(2d) [1/Å]
HCNOAlSi
• H more pronounced compared to C,N,O
• Some atom types difficult to differentiate
• 2θ = 2.3◦ at 0.8Å resolution with λ = 0.02508Å
• Mott–Bethe formula: f B(s)[Å] = 0.023934(λ/Å)2Z− fX(s)
sin2 θ
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Dynamic (Multiple) Scattering
~Si~S1
o
~S2o
~S2o
Laue Conditions (accordingly~b and~c):
(~S1o−~Si) ·~a = h1
(~S2o−~S1
o) ·~a = h′
(~S2o−~Si) ·~a = h1+h′
Simplest approximation:
Iexp(h2k2l2) ∝ |Fideal(h2k2l2)+αFideal(h1k1l1)|2
• α : re–scattering of S1o
• F(h1k1l1) must be strong
• F(h′,k′, l′) must be strong
• F(h2k2l2) must be weak
⇒ affects high resolution data
• No additional reflections
• Extinction correction (EXTI) helps, albeit inappropriate (D. L. Dorset, 1992)
• Scaling compromised by multiple scattering events
• Leads to artificially low B–factors (“map sharpening”)
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3 - Electron Diffraction Instrumentation
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Medipix / Timepix Detector Family
• first hybrid pixel detector for electrons (cf. Pilatus / Eiger)
• no read–out noise
• high dynamic range
• fast read–out: non–stop sample rotation (“shutterless data collec-
tion”)
• 512x512 and 1024x1024 pixel cameras installed in Basel (and Pisa
(Prof. Mauro Gemmi) and Stockholm (Prof. Sven Hovmöller))Diffraction image from a MFI type
zeolite:
black = 0 counts
red ≥ 1 (carbon scatter + crystal sig-
nal) count
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Eiger Chip
• Developed at PSI
• 256x256 pixel test chip with 200keV instrument
• pilot experiment for improving phosphor to higher energies ≥ 300
keV
• higher read–out (up to 8kHz), much lower dead time
• Eiger not suitable for electrons? Surprisingly good results
• Next: Jungfrau and Mönch with Si, GaAs, or CdTe
75◦ rotation data from SAPO-34
zeotype on the 256x256 Eiger chip
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The Rotation Method
• Material Science: diffraction from oriented crystals
• Rotation Method: random orientation
• Standard (“Universal”) data collection mode for organic and macromolecular crystallography
• No connection between goniometer and detector: “manual” rotation leads to very inaccurate oscillation width
• First (?) applications in electron crystallography:
– Prof. Ute Kolb, Mainz — AD3DT, step motion
– Dr. Wei Wan, Stockholm — RED, beam precession + sample rotation
– Prof. Jan Pieter Abrahams — first diffraction pattern from 3D protein crystals
• Benefit from well advance integration/ scaling programs (XDS, DIALS, SAINT, evalCCD)
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The Lens System
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C3
C2
C1
Gun / Source
Objective lens
Sample
Diffraction Plane
Imaging Plane
• Lenses C1–C3 shape beam
• Crystallography: Parallel beam
• Objective lens: sets effective detector distance to back-
focal plane = diffraction mode
• C3 not present in all microscopes
Lenses cause distortions.
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Types of Distortions
Capitani, Oleynikov, Hovmöller, Mellini, A practical method to detect and correct for lens distortion in the TEM
Ultramicroscopy (2006), 106, 66–74
Pincushion Barrel Spiral Elliptical
+ Crystallography does not require high spatial resolution (spot separation)
- Distortions translate into uncertainty of cell parameters
• Beneficial: Cell parameters from e.g. X–ray powder diffraction
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4 - Example Structures
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CSD IRELOH (courtesy Novartis)
• Spacegroup P212121
• Unit Cell 8.06 10.00 17.73 90◦ 90◦ 90◦
• RX−ray = 4.4%
• Dai et al., Eur. J. Org. Chem (2010), 6928-
6937
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IRELOH Data Statistics
Resolution #Data #Theory %Complete Redundancy Mean I Mean I/s Rmerge Rsigma
Inf - 3.65 21 30 70.0 3.30 230.86 12.70 0.1328 0.0850
3.65 - 2.40 46 55 83.6 4.60 96.03 12.78 0.1027 0.0814
2.40 - 1.89 67 77 87.0 5.18 43.24 12.13 0.1231 0.0856
------------------------------------------------------------------------------
0.91 - 0.81 317 469 67.6 1.74 1.38 1.78 0.4044 0.6805
Inf - 0.81 1347 1660 81.1 4.39 15.84 6.44 0.1604 0.1144
• Data merged from 3 crystals
• R1 = 15.52%,Rcomplete = 18.48% for 1334 reflections
• GooF = 1.42
• 195 parameters, 156 restraints (RIGU)
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IRELOH Model Building
• SHELXT distinguishes two O correctly from
the C
• Q-peaks at plausible hydrogen atom posi-
tions
• SHELX finds all but one H atoms
• C=C double bond detected
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CSD EPICZA (courtesy Novartis)
• Spacegroup P212121
• Unit Cell 11.35 12.71 13.03 90◦ 90◦ 90◦
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EPICZA Data Statistics
Resolution #Data #Theory %Complete Redundancy Mean I Mean I/s Rmerge Rsigma
Inf - 3.96 28 31 90.3 6.35 2275.45 12.58 0.0810 0.0774
3.96 - 2.50 63 65 96.9 9.58 965.01 14.68 0.1237 0.0656
2.50 - 1.91 93 94 98.9 10.46 747.39 14.67 0.1238 0.0675
------------------------------------------------------------------------------
0.91 - 0.81 345 606 56.9 1.67 20.89 2.13 0.3813 0.6147
Inf - 0.81 1819 2129 85.4 7.70 199.80 7.79 0.2275 0.1018
• Data merged from 7 crystals
• R1 = 20.2%,Rcomplete = 23.4% for 1809 reflections
• GooF = 1.55
• 259 parameters, 267 restraints (RIGU)
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EPICZA Model Building
• SHELXT misses only one O for a C
• Q-peaks at plausible hydrogen atom posi-
tions
• noisy Q–peaks
• Water molecule remains un-
detected
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Thermolysin
• Spacegroup P6122
• Unit Cell 92.5 92.5 130.9 90◦ 90◦ 120◦
• PDB ID 4N5P: dmin = 1.25Å
• Mol. Repl. with ≈ 75% completeness,
dmin = 3.5Å
• refinement (refmac) with
– MAPC FREE EXCLUDE to reduce
model bias
– SOURCE ELECTRON MB for electron
scattering factors
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Garnet Andradite Ca3Fe23+(SiO4)3
• Radiation hard
• 2 grids courtesy Sven Hovmöller’s group (Stockholm)
• Space group Ia3d, a = 12.06314(1)Å (ICSD No. 187908)
• dmin = 0.34Å (1.47Å−1
)
• Multiplicity > 14
Diffraction Geometry depends on Spot positions, but
not on intensities:
Instrument Calibration from garnet diffraction pattern
• Detector Distance
• Look-up tables for lens distortion
• Suitable for systems with comparable unit cell
volume V ≈ 1800Å3
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5 - Summary: Electron Crystallography for non–Material Scientists
Sample Prep Instrumentation Prozessing Analysis
+ from Powder
- from Solution
- Data sets / day
++ Detector∗
- Rotn Axis∗
- Lenses
- Crystal Orientn†
+ Integration
- Param. Stability
+/- Scaling
++ Direct Methods
+ Molec. Repl.
+ Refinement
- Potential Repr.
∗ Ongoing project† Project started
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6 - Acknowledgements
• Prof. J. P. Abrahams and group members at PSI / C-CINA, Basel
• Novartis (Compounds)
• Prof. Jeroen van Bokhoven, ETH Zürich
• Dr. Igor Nederlof, ASI (Medipix / Timepix)
• Dr. Bernd Schmitt, PSI Detector group (Eiger)
• Prof. Sven Hovmöller, University Stockholem (Garnet)
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