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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Organic and macromolecular crystallography with electrons
Bioinformatics and X–Ray Structural Analysis — Universität Konstanz29th January 2019
Dr. Tim Grünedes. Head of the Centre for X-ray Structure AnalysisFaculty of ChemistryUniversity of Vienna
Universität Konstanz 29th January 2019 1/0
Tim Grüne Electron Crystallography nanoArgovia A3EDPI
1 - Outline
1. Structure Determination with Crystallography
2. Electron and X–rays
3. Applications for Electron Crystallography
4. Practical Aspects
5. Radiation Damage
6. Dynamic Scattering
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
2 - Structure Determination by Single Crystal Diffraction
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
The Crystallographic Experiment
Detector
Crystal
SoRadiation
source
Planar
wave
S i
Rotation Axis of Crystal
(Diffr
action)
2θ
Data Noise (ignored)
• Diffraction spots: interaction between wave and crystal
• Experimental result: Position and Intensity for each spot
• Data recorded as frames with an area detector
• Generally true for all radiation sources, X-ray, neutron, electron
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Spot Position
• Spots positions according to Laue Conditions and orientation of Unit Cell:
(~So−~Si).~a = h
and (~So−~Si).~b = k
and (~So−~Si).~c = l
• Monochhromatic wave: ~S = (~So−~Si) depends on wavelength λ and experimental geometry
• Spot position ⇔ Crystal lattice, independent from radiation type
• Resolution dhkl of a spot from position on detector via Bragg’s law, λ = 2dhkl sin(θ)
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Spot Intensity
• Spots intensity depends on physics of interaction
X–rays interact with electrons, crystallographic map corresponds to electron density (number of electronper Volum, e−/A3).
Electrons interact with electrostatic potential from electrons + nucleus (ϕ(~r))
Neutrons interact with nucleus via weak interaction, and magnetic moment.
• Spot intensity ⇔ Unit cell content: where are the atoms, what type of atoms are they
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Types of Radiation — X–rays
Annual Growth of the PDB (X–ray)
• most advanced (pipelines from data collection to structure refinement)
• typical wavelength: λ =0.8–1.9Å
• standard structure determination
PDB CSD(Macromolecules) (Organic Compounds)
X-ray 132,000 950,000neutron 152 1,500electron 111 ?
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Types of Radiation — neutrons
PDB ID 2ZOI: D/H exchange in β–strand
(Gruene et al, J. Appl. Cryst. 47 (2014), 462–466)
1. visualisation of hydrogen atoms
2. adjacent elements (e.g. K+ vs. Cl−, Zn2+ vs. Cu+)
3. requires large crystals (≥ 1mm3)
4. (virtually) no radiation damage
5. structure determination from radiation sensitive samples (Photosystem II)
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Types of Radiation — electrons
1. strong interaction compared with X–rays: good for very smallcrystals (≪ 1µm thickness)
2. typical wavelength: 200keV = 0.0251Å
3. electron optics: imaging and diffraction with the same instru-ment
Diffraction of ZSM5 zeolite catalysts
Gruene et al., ChemEurJ (2017)
DOI: 10.1002/chem.201704213
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
3 - Electron Diffraction
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Small Crystals
Main advantage for electron crystallography:Diffraction from very small crystal (< 1µm)
Some instruments provide 5–10 nm beam diameter
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Crystallisation of Proteins
Luft, Wolfley, Snell, Crystal Growth& Design (2011), 11, 651–663
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Drops viewed through TEM
Stevenson,. . . , Calero, PNAS (2014) 111, 8470–8475 / Calero, . . . , Snell, Acta Cryst (2014) F70, 993–1008
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Electron Diffraction: Nanocrystals
organic compound
sucrose (ETH coffee bar)
Silicalite–1 / ZSM–5 (Teng Li)Gruene et al., Chem. EurJ (2018), 24, 2384–2388
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How small is “nano”?
typical protein crystal size for X-rays
typical protein crystal size for elec-trons, 100x140x1,700 nm3
volumes compare like 1m3 or 6 bath tubs of water vs. 10µl
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Effects of Crystal Volume on Diffraction Data
Reducing crystal volume reduces the resolution by (at least) two effects:
1. I(hkl) ∝ Vcrystal: 1/10 volume = 1/10 intensity
2. Henderson / Garman limit: maximum dose per volume before resolution is halved: 1/10 volume = 1/10 dosebefore radiation damage destroys crystal
From (1): In order to record the same quality diffraction pattern from a 10 times smaller crystal requires 10 timesmore intense beam.
From (1)+(2): This makes the crystal die 100 times faster
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4 - Instruments for Electron Diffraction
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Electron Microscopes
(Wikipedia)
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
The Lens System
see e.g. Zuo & Spence, “Advanced Transmission Electron Microscopy”, SpringerCarter & Williams, “Transmission Electron Microscopy”, Springer
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
backfocal plane = diffraction mode• C3 not present in all microscopes
Lenses cause distortions.
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Electron Microscope: Imaging Mode
Plane Wave Object Lense Image Plane (Detector)
Rays of equal origin focus on detector
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Electron Microscope: Imaging Mode
Plane Wave Object Lense Image Plane (Detector)
Detector noise and ra-diation senstivity re-quire low contrast im-ages
Martinez-Rucobo et al.Molecular Cell (2015) 58,1079–1089
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Electron Microscope: Diffraction Mode
Plane Wave Object Lense
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Electron Microscope: Diffraction Mode
Plane Wave Object Lense
Backfocal Plane
Rays of equal direction focus on detector
“Image” corresponds to Fourier transform of object
Image at Backfocal Plane =‖Fouriertransform of object‖
If object = crystal:
diffraction spots according to Laue conditions
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Detectors for X-rays suitable for electrons
• Eiger X 1M designed for X-ray Synchrotron radiation• 1030x1065 pixel, 75×75 µm2
• up to ν = 3kHz frame rate: data collection at synchro-tron speed
• 3µs dead time: shutterless data collection• ≤ 200keV : no radiation damage, no beam stop• 16 or 32 bit image depth & 2.8 · 10
6 ctss·pixel: high dy-
namic range
Diffraction pattern and structure for SAPO–34Tinti et al., “Electron crystallography with the EIGER de-tector”, IUCrJ (2018). 5, 190–199
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Installation of the EIGER X 1M in 1/2 day (C. Zaubitzer, ScopeM, ETH)
Removal of the previouscamera
Mounting of the EIGER X1M with adapter flange
Shielding and radi-ation monitoring
• Final shielding after 1/2 day• Vacuum OK: next morning
• Return to original state: 1 day• Gatan camera back with auto-justage
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
5 - Data Collection: Experimental Parameters
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
The Geometry Section in XDS Input File
EIGER detector not connected to the electron microscope: experimental are not present in file header.
Wavelength λ ∝ 1/E; 200keV =̂0.02508Å
✦❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂ ●❊❖▼❊❚❘■❈❆▲ P❆❘❆▼❊❚❊❘❙ ❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂❂
❖❘●❳❂ ✺✶✺ ❖❘●❨❂ ✻✷✷ ✦❉❡t❡❝t♦r ♦r✐❣✐♥ ✭♣✐①❡❧s✮✳ ❖❘●❳❂◆❳✴✷❀ ❖❘●❨❂◆❨✴✷
❉❊❚❊❈❚❖❘❴❉■❙❚❆◆❈❊❂ ✹✾✺ ✦✭♠♠✮
❘❖❚❆❚■❖◆❴❆❳■❙❂ ✲✵✳✼✸✽✺✹✺ ✲✵✳✻✼✹✶✾✺ ✵✳✵✵✸✺✻✵
❖❙❈■▲▲❆❚■❖◆❴❘❆◆●❊❂✵✳✵✷✾✺✾✸ ✦❞❡❣r❡❡s ✭❃✵✮
❳✲❘❆❨❴❲❆❱❊▲❊◆●❚❍❂✵✳✵✷✺✵✽ ✦❆♥❣str♦❡♠
■◆❈■❉❊◆❚❴❇❊❆▼❴❉■❘❊❈❚■❖◆❂✵✳✵ ✵✳✵ ✶✳✵
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
❖❘●❳✴❖❘●❨: Beam Centre
• Strong interaction of e− with crystal ⇒ direct beamweak compared with X-rays
• Direct beam visible on detector• Coordinates manually from any image viewer, e.g.
ADXV (Andy Arvai,✇✇✇✳s❝r✐♣♣s✳❡❞✉✴t❛✐♥❡r✴❛r✈❛✐✴❛❞①✈✳❤t♠❧)
• on–axis detector: direct beam ≈ ORGX/ORGY• Can vary per data set
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Detector Distance
• Detector distance set through electron optics• Detector physically not moved
• Calibration once per lens settings• Set manually with adxv and (Aluminum) powder–
pattern• Estimated accuracy: ±5mm
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
The Rotation Axis
• X-rays: rotation axis obvious from instrument setup• Electron Microscope: Objective lens determines (ef-
fective) detector distance• Image rotates step–wise depending on objective lens• powder pattern with sample oscillation (“α–wobbler):
spot on axis very strong, non–spot stays non–spot
Summed images for D560mm
Summed images for D380mm
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Determination of the Rotation Axis
• Rotation axis runs through direct beam and minimumof powder ring
• line through 2 points on rotation axis• P1 = 529/621 and P2 = 458/702
• tan(α) = ∆Y∆X
= 702−621
458−529
• ❘❖❚❆❚■❖◆ ❆❳■❙❂ cos(α); sin(α); 0
• Large radius of convergence with XDS (≈ 5−10◦)
Direction of rotation: from minimal error of spindle axis
❘❖❚❆❚■❖◆ ❆❳■❙❂ (XDS.INP) -0.6979 -0.7161 -0.0102 +0.6979 +0.7161 +0.0102❉❊❱◆ ❖❋ ❙P■◆❉▲❊ P❖❙◆ (IDXREF.LP) 0.37
◦1.01
◦
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Oscillation Width
• Current setup with “alien” detector: Sys-tem parameters not synchronized withdetector
• Step froward at C–CINA: movie duringmeasurements
dφ
dt=
∆α
∆t=
111.6◦−53.3◦
26.449s−1.264s= 2.315
◦/s
ν(EIGER) = 100Hz
⇒ ∆φ = 0.0231◦/frame
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Oscillation Width
-80-60-40-20
0 20 40 60 80
-10000 0 10000 20000 30000 40000 50000
α [d
egre
e]
t [ms]
dφ/dt [°/s]= 2.9520 ± 0.0079, α0 [°] = -62.31 ± 0.19
datafile usi (0.5*($1+$3)-t1):2
α(t) = φt + α0
• Probe α angle per 0.5s during experiment• Fit line to measurements• fast, reproducible• Oscillation width ∆φ [◦/frame] = dφ
dt/ν(EIGER)
Acknowledged: Luca Piazza. Dectris Ltd. for initial Digital Micrograph script
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
6 - Dynamic Scattering
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Dynamic Scattering
• Kinematic Theory of Diffraction: Every photon / electron / neutron scatters once in the crystal
• |Fideal(hkl)| ∝√
Iexp(hkl)
• Dynamic Scattering: Multiple Scattering events occur
• Electron Diffraction: Multiple Scattering occurs even with nanocrystals
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Dynamic Scattering
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Multiple (Dual) Scattering
~Si
~S1o
~S′o ~S2o
• First reflection ~S1o acts as source of
second reflection ~S′o.• Second reflection ~S′o overlaps with an-
other reflection ~S2o
Universität Konstanz 29th January 2019 37/0
Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Multiple (Dual) Scattering
~Si
~S1o
~S′o ~S2o
Laue Conditions (accordingly~b and~c):
(~S1o−~Si) ·~a = h1
(~S′o−~S1o) ·~a = h
′
(~S′o−~Si) ·~a = h1+h′ = h2
~S′o −~Si fulfills the Laue conditions, hence thesecondary reflection ~S′o−~S1
o overlaps with the“ordinary” reflection ~S′o−~Si.
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Experimental Treatment of Dynamic Scattering for Organic Crystals
• Exact calculation very complicated
• Not feasible for complex molecules (more than a few atoms)
• Ignorance of dynamic scattering, i.e. assumption of kinematic scattering (as in X-rays) provides reliablestructures
• Data statistics and model statistics poor, despite reliable structures
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
7 - Example Structures
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Grippostad R©, STADA
active compounds non-active compounds
paracetamol gelatineascorbic acid glycerol tristearate
caffeine lactose monohydratechlorphenamine maleate quinoline yellow (E104)
erythrosine (E127)titanium dioxide (E171)
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Single Crystal Structure from a Pharmacy Powder
1. Exp: a = 6.9, b = 9.4, c = 11.6, α = 90.6, β = 98.4, γ = 89.8
2. CSD : a = 7.1b = 9.3c = 11.7α = 90.0β = 97.7γ = 90.0; CSD search: HXACAN04, P21/n, Paracetamol,
3. Structure solved with ≤ 40% completeness
4. Difference map reveals hydrogen atoms: data sensitivity
“The existence of multiple crystal forms (polymorphs, solvates, hydrates, etc.) is playing an increas-ingly important role in establishing and protecting intellectual property rights in the pharmaceuticalindustry”
(Prof. J. Bernstein, ECM-30 (Aug. 2016), MS50-O2)
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
8 - Structure of a New Methylene Blue Derivative MBBF4
I. Regeni, Dr. J. Holstein & Prof. G. Clever, TU Dortmund
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
MBBF4 — the Crystal (J. Holstein, TU Dortmund)
C120H120B8F32N28S4Pd2, MW = 2990
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
MBBF4 — EIGER and a TEM make a Synchrotron
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
MBBF4 — Data Accuracy (J. Holstein, TU Dortmund)
Structure of MBBF4• R1 = 22.7%(2941Fo > 4σF)
• R1 = 27.2%(4832Fo)
• GooF = 1.5• 564 parameters, 1054 restraints
After addition of hydrogen atoms and re-straints: Dual conformation of BF4 becomesvisible.
Structure refinement by J. Holstein
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
9 - Electron Crystallography of Macromolecules
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Protein Crystals in the TEM
Lysozyme, ≈ 2.1Å resolution(Clabbers et el. (2017))
Thermolysin:
≈ 2×1×very thin µm3
Solvent reduces contrast(sample courtesy I. Schlichting)
Thermolysin:
about 3Å resolution
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
The Question of Resolution
e− X–ray resol. ratiodmin PDB-ID dmin PDB-ID d(e−)/d(X-rays)
Lysozyme 1.80 5K7O 0.94 1IEE 1.9Catalase 3.20 5GKN 1.50 1DGF 2.1Proteinase K 1.75 5I9S 0.83 2PWA 2.1Xylanase 2.30 5K7P 0.97 3AKQ 2.3Thaumatin 2.11 5K7Q 0.90 5X9L 2.3Trypsin 1.70 5K7R 0.75 4I8H 2.2Thermolysin 2.50 5K7T 1.12 5JVI 2.2
Liu et al., “Atomic resolution structure determination by the cryo-EM method MicroED”, Protein Science (2017), 26, 8–15
• Organic compounds reach similar resolution with electrons as with X–rays
• Proteins seem to be consistently 2× worse
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
High resolution data collection for electron Diffraction
• X-rays: Test crystals (Thaumatin, Lysozyme, . . . ) easily diffract to 1.2–1Å
• Electrons: about 2x worse so far
• Idea (I. Schlichting, K. Diederichs, independently): Combine serial crystallography with rotation method
1. Rotate sample at high dose with short lifetime but maximum resolution, e.g. 5◦ per crystal
2. Combine data from many crystals for data completness
• Very important open question in electron crystallography
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10 - Preferred Crystal Oritentation & the Missing Wedge Problem
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Missing Wedge in Electron Diffraction
Cu
Cu
140°
(a)
64°
Cu
Cu
(b)
• Crystals very often have a flat shape: always the same orientation
• Sample support stabilised by Cu-grid
• Copper grid too thick: intransparent for electrons
• Limited rotation range
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Effect of Missing Data on Map and Structure
(e) (f)
10°
(g)
30°
(h)
50°
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Complete Data from 3D Structured Grids - Coiled carbon grids
Brush Stroke causes carbon layer to coil
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
Complete Data from 3D Structured Grids - Nylon Fibres
Nylon fibres (≈ 100nm diameter) disturb preferred orientation
3D structured grids break
the preferred orientation of the crystals.
Complete data from < 5 crystals.
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Tim Grüne Electron Crystallography nanoArgovia A3EDPI
11 - Acknowledgements
• R. Pantelic (PSI/DECTRIS), S. De Carlo (DECTRIS), C. Zaubitzer (ScopeM), J. Wennmacher (PSI), J. Hol-stein (TU Dortmund), J. Heidler (PSI), A. Fecteau–LeFebvre (C–CINA), K. Goldie (C–CINA), E. Müller(PSI),S. Handschin (ScopeM), H. Stahlberg (C–CINA), N. Blanc (ScopeM), C. Schulze–Briese (DECTRIS)
• B. Luethi (DECTRIS), L. Wagner (DECTRIS), L. Piazza (DECTRIS), D. Mayani (DECTRIS)
• Y. K. Bahk (ETHZ), I. Regeni (TU Dortmund), T. Li (ETHZ), L.Muskalla (Uni Konstanz), A. Pinar (PSI), J.A.van Bokhoven (PSI/ETHZ), G. Clever (TU Dortmund)
• G. Santiso–Quinones (Crystallise!), G. Steinfeld (Crystallise!), R. Mezzenga (ETHZ), U. Shimanovich (Weiz-mann Inst.), I. Adrianssens Martiel (PSI), I. Schlichting (MPI Heidelberg), K. Diederichs (Uni Konstanz), J.Lübben (now Bruker AXS), M. Clabbers (Uni Stockholm)
nanoArgovia A3EDPI SNF Project 169258
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