Cryo-Electron Microscopy
Pavel Plevka


Transmission Electron Microscope
microscope transmission_electron_microscope
Electron source:
Thermal emission from heated cathode
Focussing:
Electro-magnetic Lenses
Detection:
Phosphor screen or CCD camera (former times: negative)
Vacuum!

Pro & Con of cryo-EM
•Pro
•Short wavelength => high resolution
•Strong interaction with materials => good contrast
•Electromagnetic lenses => standard optics (in contrast to X-ray crystallography)
•High intensity is easy to produce
•Inner structure of biomolecules is accessible
•
•Con
•High vacuum requires special treatment of sample
•Sample has to be thin to avoid 100% absorption
•Electron beam damages biological samples => short measurements => low contrast of biomolecules
•
•







Rayleigh criterion



Why electrons?
Spektrum
Visible Light:
l = 400 – 600 nm
Electrons:
l = 0.002 – 0.004 nm

Sample Preparation - Staining
ÞTo increase contrast: heavy atoms interact with electrons stronger than biomolecules (C, N, O, S,
P)
•
•Positive Staining
• treat sample with solution of salt like uranyl acetate, lead citrate, osmium tetraoxide – object
is black on light background
•Negative Staining
• place sample on dried film of heavy metal salt – object is light spot on black background
•Shadowing
• spray thin layer of heavy metal on sample to produce a shadow
•
•Disadvantage:
•Size of stain reduces resolution to about 20-30 Å







Alternative: Cryo-EM
• to avoid harsh staining which may change
  the structure of your sample
• stabilization of sample by rapid freezing of
  sample in liquid ethane to form vitreous ice
• into electron microscope at low
  temperatures to keep sample stable in
  hydrated state in vacuum
• thickness of ice layer as small as possible!
•
raw
Advantage:
• sample structure is unchanged
• inner structures of molecules are accessible




Types of Samples
•Periodic arrangement
• => 2D electron crystallography
• small or membrane proteins < 200 kDa
• resolution up to 2.5 Å
•
•Random arrangement
• => single particle technique
• macromolecular complexes > 200 kDa
• up to atomic resolution
•
•Large Organelles (Golgi, ER), whole cells
• => tomography
• resolution > 4 Å
bR_heptamer_big Fig_5
Ribosome
Bacteriorhodopsin







For a given sampling frequency, the maximum frequency you can accurately represent without aliasing
is the Nyquist frequency, which equals one-half the sampling frequency, as shown by the following
equation.




Macromolecules in water / vitreous ice are phase objects
























Signal to noise ratio



Classification and averaging
(principal component analysis)


3D Reconstruction
When the angles between the different classes are known (estimated), a 3D model can be calculated.
EM_1
Iterative Process:
3D model is used to generate 2D images which are fed into statistical analysis of images (alignment
and classification).

And then?
Try to interpret 3D map,
e.g. try to fit known crystal structures into electron density map

Cryo TEM sample preparation using Vitrobot




phi812_scheme_1.png phi812_scheme_2.png phi812_scheme_3.png












test



Picornavirus replication cycle
life_cycle_with_questions_3.jpg

… aspiring to answer basic biological questions about picornavirus life cycle. It is well
established that picornaviruses enter cells by receptor mediated endocytosis. However, it is not
known how is the picornavirus ssRNA genome delivered from virions across the membrane of the
endosome into cell cytoplasm. That is our first research question.
   When in the cytoplasm the genome is translated into a polyprotein that is then cleaved into
functional subunits. The viral proteins induce formation of so called virus-replication factories
that contain lipid vesicles derived from intra-cellular membranes, viral and cellular proteins,
ribosomes and viral RNAs. We will use modern cryo-electron tomography approaches to characterize
the factories on molecular level.
   In addition we will structurally characterize picornavirus genome replication and recombination
– processes that happen in the vicinity of the replication factories.
   Finally, we will determine picornavirus virion assembly mechanism.




1. Ktere z nize uvedenych zareni a castic interaguje nejsilneji s biologickym materialem?
a) elektrony
b) viditelne svetlo
c) paprsky X

2. Jake je v soucasnosti nejvyssi rozliseni dosazene pri studiu makromolekul pomoci
kryo-elektronove mikroskopie?
a) 1.0 Å
b) 0.23 μm
c) 0.5 nm

3. Jake je nejvyssi mozne rozliseni obrazku, ktery ma velikost pixelu 1.1Å?
a) 1.1 Å
b) 2.2 Å
c) 3.3 Å