electron microscopy: new...
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Electron microscopy: new opportunities
Helene Malet Electron microscopy group, IBS
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Electron microscopy in structural biology: Which objects can be analysed
0 50kDa 100kDa 500kDa 1MDa 10MDa 1GDa NMR
Crystallography
Single particle cryo-electron microscopy/tomography
Cellular EM/tomography
Method combinaison to study a biological process
Single particle negative stain electron microscopy
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What is negative stain electron microscopy ?
Sample preparation
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Ø The sample will be thin so electrons will be able to go through.
Ø We will see the stain as it is made of heavy atom which will interact with electrons.
Ø The stain will be more present where the protein is not: “negative stain”
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Ø The sample will be thin so electrons will be able to go through.
Ø We will see the stain as it is made of heavy atom which will interact with electrons.
Ø The stain will be more present where the protein is not: “negative stain”
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Advantages/drawbacks
Ø Advantages: – Fast – Small amount of protein (concentration around
0.01-0.1mg/ml, few ul) – Small proteins visible
(>50-100kDa) – High contrast
Ø Drawbacks: – Flattening and drying of
the sample. – Artifacts due to the stain.
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What we see in negative stain
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Single particles visualized by negative stain EM: examples
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Homogeneous (quite) Heterogeneous
150 kDa
Ø Quality control
What is negative stain EM used for ?
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1 band on gel, 2 size à 2 oligomerisation state
After fine gel filtration à Crystal of the 2 types
Ø Define oligomerization states
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• Scratch the crystal with a potter so that it becomes thin enough for the beam to go through.
• Image the small crystals obtained in the microscope in order to see the crystal lattice
Ø Organization of the protein in the crystal
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Native Fragment 56°C Fragment 37°C
Crystal
Short bacteriophage T4 fiber
Crystal packing
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Ø 3D reconstruction
Negative stain-EM Envelope of the molecule, resolution
limited to 15-20 A
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Resolution: what do we
see ?
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Resolution: what do we see ?
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Real object to be visualized Object seen in negative stain
So, negative stain EM can give important information quickly, but: • the object is not in its native environment • Can be flattened • resolution is limited to 15 A.
Can we do better ??
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Cryo-electron microscopy !
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H2O - liquid Hexagonal ice
Cubic ice Amorphous ice
Slow freezing
Rapid freezing Heat
Heat
Ø We need to freeze our sample as a thin layer: ü to resist to the vacuum ü to allow electrons to be transmitted
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a) hexagonal b) cubic c) vitrified
Dubochet et al., (1982)
a)
b) c)
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Grids used for cryo-EM
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Carbon support
Randomly oriented particles
Vitrified ice
A frozen grid
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Cryo-EM: advantages/drawbacks
Ø Advantages: – Native state – Higher resolution – Small amount of
protein (1 grid = 4 ul at 0.1-1mg/ml)
Ø Disadvantages: – Low contrast – Highly sensitive to
radiations – Minimal size limit:
250 kDa – Difficult
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Negative stain vs. Cryo-EM
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Cryo-EM: native state, so atomic resolution can (in theory) be reached!
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For high-resolution, you will need to collect on a state-of-the art electron microscope
FEI Polara FEI Titan Krios
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Electron source
Ø Purpose: generation of electrons that can be accelerated by high tension to obtain the illuminating electron beam
Thermionic gun: W or LaB6 Electrons come out when the emitter is heated
Field emission gun
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Detection
Ø On films that need to be developed and digitized
Ø On CCD camera
Ø On direct detector
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For high-resolution, you will need to do a lot of image processing
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+
Combination EM-crystallography
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Or… high resolution EM
Zhang et al, Cell, 2010
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Ø Smaller complexes less and less symetric
Ribosome (no symmetry, 4MDa, 4 Å resolution)
Proteasome (D7 symmetry, 700kDa)
Li et al, Nature methods, 2013 Bai et al, eLife, 2013
Or… high resolution EM
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Towards high resolution of smaller complexes
Liao et al, Nature, 2013 Lyumkis et al, Science, 2013
Crystallography
EM
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Merk et al., Subramaniam, Cell 2016
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The dream: understanding a system from the cell context to the atomic level
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If complexes heterogeneous: separation of different populations within the same sample Ø Different 3D volumes with particles chosen randomly. Ø Competitive alignment to determine to which structure
each particle belongs
Simonetti et al, Nature 2008
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If complexes heterogenous: separation of time-resolved states
Fischer et al, Nature 2010