Title: Cryogenic Transmission Electron

Microscopy

Name: Sana Azam

Reg No.: 2023-mphil-1693
Course Title: Enzyme Biochemistry
Submitted To: Prof. Dr. Muhammad Tayyab

Institute of Biochemistry and Biotechnology

University of Veterinary and Animal Sciences - UVAS
Syed Abdul Qadir Jillani (Out Fall) Road, Lahore - Pakistan
Cryogenic Electron Microscopy (Cryo-EM):
Cryogenic Electron Microscopy (Cryo-EM) is a cutting-edge
structural biology technique that allows scientists to visualize the
three-dimensional (3D) structures of biological macromolecules,
cellular components, and large protein complexes at high resolution.
It has revolutionized structural biology and has become a powerful
tool for understanding the architecture and function of complex
biological systems. Here's a detailed overview of Cryo-EM:

Principle of Cryo-EM:
Cryo-EM is based on the principles of transmission electron
microscopy (TEM), which uses a beam of electrons to image the
specimen. However, Cryo-EM is unique in that it images specimens
at cryogenic (ultracold) temperatures, typically around -196 degrees
Celsius (-321 degrees Fahrenheit). The low temperature helps
preserve the biological sample in a near-native, hydrated state and
minimizes radiation damage from the electron beam.

Cyro Electron Microscope

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Working
 Sample Preparation: The process begins with preparing a thin
layer of the specimen ‘ in a buffer solution. A small droplet of
this sample is applied to a specially designed electron
microscopy grid coated with a thin layer of amorphous carbon.
The excess solution is blotted away, leaving behind a thin film
of the specimen.
 Freezing: The grid with the specimen is rapidly plunged into a
cryogen (usually liquid ethane or liquid nitrogen) at cryogenic
temperatures. This process vitrifies the sample, turning it into a
non-crystalline, glass-like state that traps water molecules
without forming ice crystals.
 Imaging: The cryogenically frozen grid is inserted into a
transmission electron microscope. A focused beam of electrons
is transmitted through the specimen, and the resulting electron
micrographs are recorded by a detector. The recorded images
are two-dimensional projections of the 3D structure of the
specimen.

Work flow of cyro electron microscope

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 Data Collection: Multiple micrographs are collected from
various orientations of the specimen by tilting the grid or the
stage of the microscope. This produces a set of two-dimensional
images that capture different views of the sample.
 Image Processing: Advanced computational techniques are
used to process the collected images. This includes aligning and
averaging the images to enhance signal-to-noise ratios and to
correct for distortions and artifacts.
 3D Reconstruction: The processed 2D images are used to
generate a 3D reconstruction of the specimen's structure. This is
typically done using computational methods such as Fourier
transformation and back-projection. The final 3D model reveals
the atomic positions and structural details of the biological
specimen.

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Advantages of Cryo-EM:
 Near-Native Conditions: Cryo-EM allows the study of
biological samples in a close-to-native state, preserving their
natural structures and conformations.
 High Resolution: Recent advancements in Cryo-EM have
pushed its resolution to near-atomic levels, making it possible to
visualize molecular details and interactions.
 No Need for Crystallization: Unlike X-ray crystallography,
Cryo-EM does not require the crystallization of samples,
making it suitable for large and flexible macromolecular
complexes.
 Versatility: Cryo-EM can be applied to a wide range of
biological specimens, including proteins, nucleic acids, viruses,
cells, and cellular organelles.
 Dynamic Information: It can capture dynamic processes by
studying multiple snapshots of a specimen, revealing
conformational changes and intermediate states.

Disadvantages
 Expense and Accessibility:Cryo-EM equipment is expensive to
purchase, maintain, and operate. This limits its accessibility for
smaller research laboratories with budget constraints.
 Complexity of Sample Preparation:Preparing samples for Cryo-
EM requires specialized skills. The process involves rapidly
freezing the sample in a way that avoids ice crystal formation,
which can be technically challenging.
 Sample Heterogeneity: Biological samples can be
heterogeneous, meaning that they exist in multiple
conformations or states.

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Applications of Cryo-EM:
Cryo-EM has diverse applications across various scientific
disciplines:

 Structural Biology: Determining the structures of biological

macromolecules, including proteins, nucleic acids, and
complexes, to understand their functions.
 Drug Discovery: Visualizing the structures of drug targets and
their interactions with potential drug compounds.
 Virology: Studying the structures of viruses and their
components to aid in vaccine development and antiviral drug
design.
 Cell Biology: Investigating the structures of cellular organelles
and complexes to understand cellular processes.
 Neuroscience: Mapping the structures of neuronal connections
and synapses to study the brain's functional circuits.
 Materials Science: Characterizing nanomaterials and
nanoparticles for various applications.
 Cryo-EM has transformed our ability to explore the molecular
world with unprecedented detail and is at the forefront of
structural biology, enabling groundbreaking discoveries in
multiple scientific fields.