Structural Chemistry and Structural Biology with Electrons · Tim Grüne IAC Kolloquium Göttingen...

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

12th December 2016 Structural Chemistry with Electrons 1/27


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)



Single Organic Crystals from Powder

Novartis IRELOH:

Ø= 1,700nm = 1.7µm

Novartis EPICZA:

Ø= 500nm = 0.5µm

Novartis EPICZA:

Ø= 650nm = 0.65µm



Zeolite Crystals at their Working Size

≈ 300nm Si-only zeolite type MFI (Pnma, 20.1Å, 19.7Å, 13.1Å)



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



2 - Electrons as Radiation Source



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



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 (?)



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



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”)



3 - Electron Diffraction Instrumentation



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



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



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)



The Lens System

��

��

��

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.



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



4 - Example Structures



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



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)



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



CSD EPICZA (courtesy Novartis)


• Unit Cell 11.35 12.71 13.03 90◦ 90◦ 90◦



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)



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



Thermolysin


• 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



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



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



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)


Structural Chemistry and Structural Biology with Electrons · Tim Grüne IAC Kolloquium Göttingen...

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