This document discusses cryo-electron microscopy (cryo-EM), a technique used to image biological macromolecules in their native frozen state without staining or chemical fixation. It describes how cryo-EM overcomes limitations of traditional transmission electron microscopy by rapidly freezing samples to vitrify water and prevent ice crystal formation. The document outlines key developments in cryo-EM including contributions from Richard Henderson, Jacques Dubochet, and Joachim Frank who were awarded the 2017 Nobel Prize in Chemistry. Examples of structures solved using cryo-EM such as the Zika virus and nuclear pore complex are provided. Advantages and applications of cryo-EM are summarized.

1. By,
Aiswariya S,
Akash Mahadev Iyer &
Lifa Prathapan
S M.Sc Biochemistry
Department of Biochemistry
University of Kerala

2. Is the human
body made
up of cells or
molecules or
atoms?

3. Does knowing the structure of a molecule matters?
As Francis Crick, one of Britain's great scientists, once said:
"If you want to understand function, study structure."
Molecular structure - key to understanding Nature's intricate design
mechanisms and blueprints.
Chemical structure determines the molecular geometry and physical
properties of a compound by portraying the spatial arrangement of
atoms and chemical bonds in the molecule.
Important visual representation of a chemical formula- Elemental
composition
Higher resolution instrumentation allows us to study single molecules
For eg; Chemical structure of amino acids influences three
dimensional structures of proteins..

4. Structural biology explores the 3-D shapes of biological
macromolecules and their complexes at atomic
resolution.
Approaches such as X-ray crystallography, NMR, ESR,
Electron microscopy etc. are used to determine the
myriad physical forms that proteins and nucleic acids
adopt

5. Let’s compare them…

7. a)Rhodospirillum rubrum in light microscope
b)A thin section of R.rubrum in TEM
Scanning Tunneling
Microscopic Image of DNA
double helix
Membrane protein Aquaporin visualized by Atomic Force Microscopy

8. Characteristic Light Microscope Electron microscope
Useful magnification 2000 X 500,000 X
Maximum resolution 200 nm 0.2 nm
Image produced by Visible light rays Beam of electrons
Image focused by
Glass objective lens Electromagnetic objective
lens
Interior Air filled Vaccum
Radiation source
Mercury Lamps, Xenon
Lamps, Lasers or Light-
emitting Diodes
High voltage Tungsten
Lmp (50kV)
Object Alive or Dead Dead only
Coloured images produced Yes No, only black and white
Use
Highly detailed images,
3D surface imaging, Sub-
cellular visualization,
Virus
Reasonable details-
morphology. Visualize
small invertebrates, whole
cells etc.
Cost
Usually between $150-
$15,000
More than $50,000

9. Electron Microscopes-Types
Transmission electron microscopy (TEM) -electrons that
are transmitted through the ultra-thin sample
 2-D images
 Provide details about their internal composition.
Scanning Electron Microscopy (SEM) - electrons that are
scattered from sample
 3D image
 Samples that are used for SEM need to be electrically
conductive -biological samples need special
preparation.
 They must be fixed, dried, and coated with a thin layer
of a conducting material.

10. Instrumentation
TEM parts
Electron column
with specimen
chamber- BODY
Electron source
Thermionic el-
gun
Field
emission gunSeveral lenses
that focus el
beam
Imaging system
Auxiliary
components
Vacuum system
High vacuum
minimizes collision
frequency of electrons
with gas atoms and
ensures stable
operation
Eg; LaB6
crystal,
Tungsten
filament
Sharp
pointed
cathode

11. Sample
Primary Backscattered electrons
Atomic number and
Topographical information
Cathodoluminescence
Electrical information
Secondary electrons
Topographical Information
X- rays
Thickness
Composition information
Auger electrons
Surface sensitive
Compositional information
Incident Beam

12.  Lenses in electron
microscopes are grouped
into three systems ;
The condenser lens - directs the
el- beam into the sample
 Controls the beam intensity and
beam coherence
The objective lens - produces a
magnified real image of the
sample and is used for focusing.
The projector lens -allow easier
control of the magnification and
projects the magnified image onto
a detector.

13. Detectors
• Several types of detectors can be used in TEM .
• Detectors use the information contained in the electron
waves exiting from the sample to form an image.
• CCDs
• Hybrid pixel detectors
• MAPS-Monolithic Active Pixel Sensors
• DEDs-Direct Electron Detectors

14. In CCD detectors, electrons are
converted to photons upon hitting
scintillating plate, and photons are
recorded by CCDs detectors.
Recorded signal had a poor signal-
to-noise ratio due to scattering of
electrons on the scintillating plate
DEDs -directly acquire
electrons without the need of
converting them to photons-
Improved resolution, better
signal-to-noise ratio, faster
readout.

15. Utility of TEM
use a beam of electrons
to examine the structures of molecules and materials at the atomic
scale.
As the beam passes through very thin sample, it interacts with the
molecules, which projects an image of the sample onto the detector
(CCD).
-thus, it can reveal much finer details than light
microscopy.
Biomolecules – incompatible with the high-vacuum conditions and
intense electron beams used in traditional TEMs.
High energy electrons cause severe radiation damage to delicate
samples by breaking the atomic bonds, loss of water and secondary
damage that is caused by the radicals that are generated.

16. What is this all about?
Praised by Nature as its 2015 ‘Method of the Year’
Cryoelectron microscopy is a method for imaging frozen-hydrated
specimens at cryogenic temperatures by electron microscopy.
Specimens remain in their native state without the need for dyes or
fixatives, allowing the study of fine cellular structures, viruses and
protein complexes at molecular resolution.

17. Why use Cryo EM???
Cryo-EM uses frozen
samples, gentler
electron beams and
sophisticated image
processing to
overcome many of the
problems faced by
TEMs.
Native State of Sample
Vitrified Water
No Staining
Automated 3D
Reconstruction
Validated Structures

18. • A trio of scientists share the 2017 Nobel prize in
chemistry - for developing Cryo-electron microscopy:
(from left) Jacques Dubochet, Joachim Frank and Richard
Henderson.

19. The electron microscope’s resolution has radically improved in the last
few years, from mostly showing shapeless blobs to now being able to
visualise proteins at atomic resolution…

20. Steps in Cryo EM
SAMPLE PREPARATION
DATA COLLECTION and
ANALYSIS

21. Cryo-EM samples -purified to perfect homogeneity
Heterogeneity- assessed by SDS-PAGE and size-
exclusion chromatography.
Native gel electrophoresis and static and DLS techniques
can also be used to assess the polydispersity
(characterized as particles of varied sizes in the
dispersed phase) of more complex samples.
The best way to assess quality of sample -directly
visualize it by EM.
All samples for cryo-EM are first analyzed by negative
staining electron microscopy.
SAMPLE PREPARATION

22.  Negative-stain EM images provide -information on a sample-
presence of contaminants or aggregates, size, shape etc.
Three major steps:
Adsorption of the sample on carbon-coated grid,
Blotting the excess sample and washing the grid with deionized
water, and
Staining with a heavy metal solution
 Heavy metal stains- uranyl acetate, phosphotungstic acid,
ammonium molybdate, methylamine vanadate.
 Heavy ions -readily interact with the electron beam and produce
phase contrast.

23. Vitrification- Ice free solidification of an aqueous solution
Particles are kept in a native state by embedding them in a thin layer
of vitrified water- very weak contrast.
Imaging at cryo temperatures- reduces specimen movement and
radiation damage.
Quick freezing solidifies the water without allowing ice crystals to
form (ice crystal formation damages biological molecules and
generate strong contrast that obscures molecular features.)
Vitrification

24. Vitrification
Liquid nitrogen has a significantly lower
boiling point (-196o C) compared to liquid
ethane (-88.6o C).

25. When sucrose is cooled slowly it results in
crystal sugar (or rock candy), but when cooled
rapidly it can form syrupy cotton candy
(candyfloss).

26. Nitrogen is not directly used; It can
make crystals due to slow cooling!!

29. 1)
2)
Summarize….

30. 3)
4) 5)

31. v
6)
7) 8)

32. 9)
10)

33. 11)
12)

34. 13)

35. 14.a) 14.b)

36. 14.c)

37. 14.d)

38. • Richard Henderson –was first to actually figure out the 3D structure
of a protein using an electron microscope.
• Jacques Dubochet found a way of freezing aqueous samples fast
enough to preserve molecules’ shape in a glassy ice matrix.
• Joachim Frank’s major contribution to the field was in processing
and analysing cryo-EM images.
• Developed computational methods for taking images of multiple,
randomly-oriented proteins and compiling them into sets of similar
orientations to obtain sharper 2D images- 3D image could be
constructed from these 2D projections.

39. It was James Dubochet who put the ‘cryo’ into cryo-EM.
He developed a method for freezing water so rapidly that the water
forms a disordered glass, rather than crystalline ice.
This is important because ordered ice crystals would strongly
diffract the microscope’s electron beam, obscuring any information
about the molecules being studied.
Catapulting the sample into a bath of liquid nitrogen-cooled ethane
freezes the thin film of water on a TEM sample so quickly that the
water molecules don’t have time to arrange into a crystalline lattice.
In this ‘vitrified’ sample, the water is disordered but the 3D structure
of the biomolecules in the sample is retained

41. Spliceosome
Spliceosome-cellular
machine that performs
pruning of mRNA.
Chops out unnecessary
pieces, called introns,
and joins together the
leftover, essential
sequences, called exons.
The edited mRNA is then
exported to the cell’s
cytoplasm, where it gets
translated into protein.

42. The structure of the
Zika virus was solved
in just a few months
using cryo-EM.
Searching for
possible sites that
could be targeted by
drugs to prevent the
spread of the virus
The Zika virus

43. TRPV6 protein situated in brush border of enterocytes- acts as
Calcium channel
Deviations in TRPV6 channels - advancement of malignancy by
upsetting the control of cell multiplication and cell demise.
Drug development to correct abnormalities in calcium uptake, which
have been linked to cancers of the breast, endometrium, prostate,
and colon.
Scientists used advanced
cryo-electron microscopy
to image TRPV6.

44. Gatekeeper of the cell’s nucleus- the nuclear
pore complex (NPC) controls the shuttling of
thousands of different proteins, RNA molecules,
and nutrients between the nucleus and
surrounding cytoplasm.

45. Advantages
Allows the observation of specimens that have not been
stained or fixed in any way.
Showing them in their native environment.
Less in functionally irrelevant conformational changes.
Disadvantages
Expensive.
The resolution of cryo- electron microscopy maps is not high
enough

46. Applications
• Studying the Molecular mechanisms of various life
processes
• Cryo-EM is used to characterize the structure and
mechanism of action of macromolecular complexes, -
spliceosome and snRNP complexes, anaphase-
promoting complex, ion channels, and human γ-
secretase.
• Nano particle research
• Structure based Drug design
• Material Characterization

47.  ’Cryo-electron microscopy of vitrified specimens.’ -Dubochet J, Adrian M, Chang JJ,
McDowall AW, Schultz PQuarterly Reviews of Biophysics.
 Methods in Molecular Biophysics- By Nathan R. Zaccai, Igor N. Serdyuk, Joseph Zaccai,
Cambridge University Press
 ‘Cryo electron microscopy to determine the structure of macromolecular complexes’-
Marta Carroni and Helen R. Saibil- Methods - Journal – Elsevier.
 ‘Cryo-electron tomography: The challenge of doing structural biology in situ’Vladan
Lučić, Alexander Rigort, Wolfgang Baumeister- Journal of Cell Biology
 ‘Introduction to high-resolution cryo-electron microscopy’- Mariusz Czarnocki-Cieciura
Marcin Nowotny- Postępy Biochemii
 ‘Electronic detectors for electron microscopy’ A.R Faruqi and R. Henderson- Current
Opinion in Structural Biology
 Biophysical Techniques in Drug Discovery- edited by Angeles Canales, Royal Society of
Chemistry.
 ‘Move over X-ray crystallography. Cryo-electron microscopy is kicking up a storm by
revealing the hidden machinery of the cell.’- Ewen Callaway, NATURE .