New EM Technologies:
unveiling the wonders of nature at the molecular level.
Dr. Sacha De Carlo
Applications Lab
FEI Company - Eindhoven
CEITEC Winter School on Structural Cell Biology
February 9-13, 2015
Limits of Resolution of Various Imaging Technologies
Currently achieved resolution
Prediction for resolution improvement
Leis, et al. (2009)
The Never-Ending Quest for Higher Resolution in EM
2015
/ NMR
Current state-of-the-art
3
Xiao-chen Bai, Greg McMullan & Sjors Scheres
2011-2012: challenges
“Smaller (<500kDa), potentially
dynamic, protein complexes are
at the heart of cell function, and
understanding how they work
will generate fundamental
insights that will be key to the
development of biomedical
therapeutics in the coming
decades.”
Milne et al. (2012) Cryo-EM: a
primer for the non-microscopist.
FEBS Journal 280
2015: >700
2015: 35
State-of-the-Art
Current and future challenges
Sample size, quality (bottleneck)
Beam Damage (dose fractionation)
Better Detectors (fast, counting)
Better Hardware (PP, aberrations)
Types of Samples Studied by Cryo-EM
70S E. coli
ribosome
Hepatitis B
virus core
Actin-myosin
filament
Light-harvesting
2D crystal
Baker and Henderson (2001)
Two-dimensional crystalsHelical symmetrySingle particles with
icosahedral or other
symmetries
Single particles
with little or no
symmetry
Sample Types
Type Examples Reconstruction
Method
Typical
resolution
Single
particles
Icosahedral viruses,
GroEL, ribosome
Single particle
reconstruction
20-3 Å
Filaments Flagella, filamentous
viruses, actin, tubular
crystals
Helical
reconstruction
15-3 Å
2D crystals BRhodopsin, aquaporins,
tubulin, S-layers
2D electron
crystallography
10-1.9 Å
Ensembles HIV capsids, whole mount
(small) cells
Electron Tomography 40-20 Å
From Sample to Structure (SPA workflow)
Slide adapted from De Carlo & Rémigy,
Biophysics in drug discovery (April 2015)
Automation = High Throughput
Single particle analysis (SPA)
11
10’s
Film scanner Digital CCD CMOS
Krios
FEG
Automation
70’s
Manual
Automation -> electronic detectors
Negative Stain vs. Vitreous Ice
Automated image acquisition software – FEI EPUTM
Potter et al. (1999). Leginon: … 1000 images a day. Ultramicroscopy 77, 153-161
?FEI Titan Krios
2,504,547; ?? Å
“The more, the better”
dogma
Direct electron detectors
The Resolution Revolution
16
Science 343 (2014)
17
Science 343 (2014)
18
Science 343 (2014)
Improving detection: “seeing electrons”
Improved Detective Quantum Efficiency (DQE)
-25°C
CCD
array
Peltier cooler
e- e- e- e- e- e-
Fiber
optics
ScintillatorScintillator
-25°C
CMOS
array
Peltier cooler
e-e-e-e-e-e-
Scintillator
Fiber Optics
Coupling
CCD chip
e- e-
PSF
CCD: multi stage conversion of electron
energy via fiber or lens optics
CMOS: direct conversion of electron energy
without fiber or lens optics
Assessing the quality of a detector
𝐷𝑄𝐸 𝑘 = 𝐷𝑄𝐸(0)
𝑀𝑇𝐹2
𝑁𝑇𝐹2
Meyer & Kirkland (2000)
De Ruijter (1995)
realistically ideal
CCD
DDD
DQE comparison for various detectors at 300kV
McMullan et al. (2014) Ultramicroscopy 147, 156-163
Assessing detector performance
Comparison tests with Gatan US-4000
CCD mounted on the F20; same FOV
and same illumination conditions
Cross-grating gold replica: Falcon2
80kX nominal mag (1.25Å pix size); Nyquist = 2.5Å
2.3Å gold reflection line seen past Nyquist !!
Cross-grating gold replica: Gatan CCD
100kX nominal mag (0.9 Å pix size); Nyquist = 1.8Å
Nothing visible besides a few Thon rings in the center
Ribosome (Krios, 300kV, Falcon2)
Falcon2
CCD
Movie mode
(aka dose fractionation)
Movie processing (Motioncorr, Relion, …)
Bai et al (2013). eLife 2
Motion correction: “movie mode”
Bai et al (2013). eLife 2
Motion correction: Rotavirus particles
DED Cameras – Gatan K2
Li et al (2013) Nature Methods 10, 584-590.
Figure 2. Motion correction restores the lost high-resolution information. (a) Fourier transforms of an image of frozen hydrated archaeal
20S proteasomes. Representative ‘near-perfect’ image in which Thon rings extend to nearly 3 Å. The cross-correlation (CC) between
image Thon rings at 5–10 Å and simulated ideal Thon rings over the same resolution range is 0.192. (b) The 24 individual subframes
used to create a were cross-correlated, and relative positional shifts were determined as described in the text and Online Methods. On
the basis of these calculations, the path of motion between the first (large black dot with arrow) and last subframes can be determined.
(c) Fourier transform of a after motion correction, where the Thon ring CC is 0.233. (d) Fourier transform of a representative imperfect
image showing a predominantly unidirectional resolution cutoff at ~20 Å. The Thon ring CC is only 0.092. (e) Trace of detected motion
between subframes used to create d. (f) Fourier transform of d after motion correction, showing that resolution has been isotropically
restored; the Thon ring CC improved to 0.238. The narrow white band was caused by residual fixed-pattern noise in each subframe,
which was subsequently eliminated.
DED Cameras – FEI Falcon 2
1-little movement
Uncorrected
Drift-corrected
3Å 3Å
3Å
DED Cameras – FEI Falcon 2
2-modest movement
Uncorrected
Drift-corrected
3Å
3Å
3.7Å
Membrane proteins:
33
TRPV1 (300 kD)
ABC-transporter (135 kD)
γ-secretase (170 kD)
Kutti R. Vinothkumar, et al. Nature (2014) 515 pp. 80-84 (Falcon)
Erhu Cao, et al. Nature (dec 2013) 504 pp. 113-118 (K2)
Maofu Liao, et al. Nature (dec 2013) 504 pp. 107-112 (K2)
JungMin Kim, et al. Nature (2014) (K2)
Joel R. Meyerson, et al. Nature (2014) (Falcon)
Peilong Lu, et al. Nature (2014) (K2)
Rouslan G. Efremov, et al. Nature (2014) (Falcon)
Glutamate receptor (460kD)
Complex I (1MD)
Ryanodine receptor (2.2MD)
Filaments:
34
Inflammasomes
Microtubules
MAVS filament
Fromm S. et al. JSB (2014) (Falcon, K2)
Lu A, et al. Cell (2014) 156 pp. 1193-1206 (Falcon)
Wu B, et al. Molecular Cell (2014) 55 pp. 511-523 (K2)
Von der Ecken J, et al. Nature (2014) (Falcon)
Galkin VE, et al. Structure (2014) (Falcon)
Alushin GM et al. Cell (2014) 157 pp. 1117-1129 (film)
F-Actin
(M)any high resolution structure(s) seems to be a
ticket for a high impact publication!
TMV
Ribosomes (2014):
35
Hussain T, et al. Cell (2014) 159 pp. 597-607 (Falcon)
Bischoff L, et al. Cell Rep. (2014) 9 pp. 469-475 (Falcon)
Arenz S, et al. Molecular Cell (2014) (Falcon)
Brown A, et al. Science (2014) 346 pp. 718-722 (Falcon)
Greber BJ, et. Al. Nature (2014) (Falcon)
Shao S, et al. Molecular Cell (2014) 55 pp. 880-890 (Falcon)
Voorhees RM, et al. Cell (2014) 157 pp. 1632-1643 (Falcon)
Wong W, et al. eLife (2014) 3 (Falcon)
Fernandez IS, Cell (2014) 157 pp. 823-831 (Falcon)
Amunts A, Science (2014) 343 pp. 1485-1489 (Falcon)
Greber BJ, et al. Nature (2014) 505 pp. 515-519 (cover) (Falcon)
Many high impact publications on ribosomal complexes! Routinely ≤4Å
Full de novo model building
Emetine drug
Electromagnetic lenses
(Spherical aberration)
Round EM lenses -> Cs (aberration)
37
Alignment accuracy in a Titan Krios
38
39
Aberration-corrected TEM
40
Aberration-corrected TEM
Aberration-corrected cryo-TEM (<3Å)
41
Data (Nature, in press) courtesy of Prof. Holger Stark, MPI Göttingen
Enhancing contrast: Phase Plate
Zernike Phase Contrast
43
Frits Zernike (1888-1966)
The basic principle to make phase changes
visible in phase contrast microscopy is to
separate the illuminating background light
from the specimen scattered light, which make
up the foreground details, and to manipulate
these differently
Zernike Phase Plate in TEM
44
Dai… & Chiu (2014) Nature Protocols 9, 2630–2642
Principle
When an electron beam passes a continuous heated carbon film:
1. The beam leaves a white footprint (underfocus)
45
hole footprint beam
2. A phase shift occurs of the central beam relative to the diffracted beam
π/2
Contrast improvement for tomography
46
• The FEI Volta Phase Plate provides “high defocus” contrast with in-focus imaging
• The FEI VPP has a long lifetime, regenerates itself, does not require an airlock nor
frequent replacements, does not need a centering mechanism and is
contamination free
• The FEI VPP is fully automated, easy and simple to use, embedded in the TEM UI
and Explore3D tomography software (version 4.1)
Danev R, et al. (2014), PNAS 111, p. 15635
With better detectors we maximize output with
minimum effort
Bai et al. (2013). Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2
?FEI Titan Krios
2,504,547; ?? Å
“The more, the better”
dogma
“4D” cryo-EM !!
Fischer et al. (2010) Nature 466, 329-333
Thank You
http://www.fei.com/life-sciences/