Electron cryo-microscopy: Using inexpensive technology to produce high- resolution images

Date:
July 13, 2020
Source:
Martin-Luther-Universita"t Halle-Wittenberg
Summary:
Biochemists have used a standard electron cryo-microscope to achieve
surprisingly good images that are on par with those taken by far
more sophisticated equipment. They have succeeded in determining
the structure of ferritin almost at the atomic level.



FULL STORY ========================================================================== Biochemists at Martin Luther University Halle-Wittenberg (MLU) have used
a standard electron cryo-microscope to achieve surprisingly good images
that are on par with those taken by far more sophisticated equipment. They
have succeeded in determining the structure of ferritin almost at the
atomic level.

Their results were published in the journal PLOS ONE.


========================================================================== Electron cryo-microscopy has become increasingly important in recent
years, especially in shedding light on protein structures. The developers
of the new technology were awarded the Nobel Prize for Chemistry in
2017. The trick: the samples are flash frozen and then bombarded with electrons. In the case of traditional electron microscopy, all of the
water is first extracted from the sample. This is necessary because the investigation takes place in a vacuum, which means water would evaporate immediately and make imaging impossible.

However, because water molecules play such an important role in
biomolecules, especially in proteins, they cannot be examined using
traditional electron microscopy. Proteins are among the most important
building blocks of cells and perform a variety of tasks. In-depth
knowledge of their structure is necessary in order to understand how
they work.

The research group led by Dr Panagiotis Kastritis, who is a group
leader at the Centre for Innovation Competence HALOmem and a junior
professor at the Institute of Biochemistry and Biotechnology at MLU,
acquired a state-of-the-art electron cryo-microscope in 2019. "There
is no other microscope like it in Halle," says Kastritis. The new
"Thermo Fisher Glacios 200 kV," financed by the Federal Ministry of
Education and Research, is not the best and most expensive microscope
of its kind. Nevertheless, Kastritis and his colleagues succeeded in determining the structure of the iron storage protein apoferritin
down to 2.7 aangstro"ms (AA), in other words, almost down to the
individual atom. One aangstro"m equals one-tenth of a nanometre. This
puts the research group in a similar league to departments with far more expensive equipment. Apoferritin is often used as a reference protein
to determine the performance levels of corresponding microscopes. Just recently, two research groups broke a new record with a resolution of
about 1.2 AA. "Such values can only be achieved using very powerful instruments, which only a handful of research groups around the world
have at their disposal. Our method is designed for microscopes found in
many laboratories," explains Kastritis.

Electron cryo-microscopes are very complex devices. "Even tiny
misalignments can render the images useless," says Kastritis. It
is important to programme them correctly and Halle has the technical
expertise to do this. But the analysis that is conducted after the data
has been collected is just as important. "The microscope produces several thousand images," explains Kastritis. Image processing programmes are
used to create a 3D structure of the molecule. In cooperation with
Professor Milton T. Stubbs from the Institute of Biochemistry and
Biotechnology at MLU, the researchers have developed a new method to
create a high-resolution model of a protein. Stubbs' research group uses
X-ray crystallography, another technique for determining the structure
of proteins, which requires the proteins to be crystallised. They were
able to combine a modified form of an image analysis technique with the
images taken with the electron cryo-microscope. This made charge states
and individual water molecules visible.

"It's an attractive method," says Kastritis. Instead of needing very
expensive microscopes, a lot of computing capacity is required, which
MLU has. Now, in addition to using X-ray crystallography, electron cryo-microscopy can be used to produce images of proteins -- especially
those that are difficult to crystallise. This enables collaboration,
both inside and outside the university, on the structural analysis of
samples with medical and biotechnological potential.


========================================================================== Story Source: Materials provided by Martin-Luther-Universita"t_Halle-Wittenberg. Note: Content may be edited
for style and length.


========================================================================== Journal Reference:
1. Farzad Hamdi, Christian Tu"ting, Dmitry A. Semchonok, Koen
M. Visscher,
Fotis L. Kyrilis, Annette Meister, Ioannis Skalidis, Lisa Schmidt,
Christoph Parthier, Milton T. Stubbs, Panagiotis L. Kastritis. 2.7
AA cryo-EM structure of vitrified M. musculus H-chain apoferritin
from a compact 200 keV cryo-microscope. PLOS ONE, 2020; 15 (5):
e0232540 DOI: 10.1371/journal.pone.0232540 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/07/200713120020.htm

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