Nature: For the first time, atomic-level structural changes in the mechanism of active translation were observed directly within cells

Every living cell relies on proteins to function, and the process of protein synthesis — translation — is essential
for survival.
Bacteria are no exception, and molecular machines involved in translation are one of the most common targets of
antibiotics.

Now, for the first time, scientists have visualized in atomic detail how antibiotics affect the process of
protein production within bacterial cells.
The study, published in the journal Nature, also marks the first time scientists have observed atomic-level structural changes in active translation mechanisms directly within cells, rather than using isolated molecules in test tubes
.
Importantly, this approach allowed them to identify the mechanisms
by which these machines "talk" to each other within the cell.

The study was conducted
in collaboration with researchers from the Max Planck Institute for Biophysical Chemistry (G?ttingen), the Wellcome Centre for Cell Biology, the University of Edinburgh and the Universit?t Berlin.
The study also includes contributions from the Zimmermann-Kogadeeva and Bork groups at the Heidelberg EMBL, who helped researchers conduct bioinformatics analyses to observe the diversity
of ribosomal proteins > 4,000 representative bacteria.

Mahamid and her team used a technique called cryo-tomography (cryo-ET) to conduct the study
.
This tiny bacterium causes SARS in humans, and despite its size being only one-ten-thousandth of a millimeter, it has a fully functioning protein synthesis mechanism
.

"We chose Mycoplasma because they are one of the smallest living cells and are widely used as model cells for systems biology and synthetic biology research," said
Liang Xue, a postdoctoral fellow with Mahamid's team and first author of the study.

Cryo-ET allows researchers to use electron microscopy to take a series of images of a fast-frozen biological sample and combine the resulting images into a three-dimensional view of the cells — a bit like a small MRI machine
.
"With large-scale cryogenic et data obtained from the original preserved cells, it is possible to capture high-resolution snapshots of molecular machines in different states of activity and combine them into a movie," Mahamid said
.

One of the most prominent structures when we look at cryogenic et-cells are tiny black spots, these are ribosomes
.
"Ribosomes are one of the oldest macromolecular machines, and it may have existed before the cells appeared," Professor Xue said
.
Ribosomes are the main molecular machinery involved in protein translation, present in all cells, from bacteria to humans
.

Mahamid's method not only allowed them to discover and count ribosomes inside bacteria, but also to see their structure
at atomic resolution.
By studying the large number of ribosomes that are "frozen" at different stages of their active cycle, scientists can decipher changes
in ribosomal structure during protein synthesis.
Not only that, but they can also locate ribosomes in three-dimensional space within cells, which allows them to identify how the translation process is spatially organized
.

Xue said, "Within living cells, ribosomes function as highly interconnected systems, not as individual molecular machines
.
We revealed novel features of ribosomes and different pathways
of translational reactions in cells.
”

On top of that, using cryogenic et, researchers can observe what
happens when antibiotics enter cells and bind to ribosomes.
For example, they can confirm that two broad-spectrum antibiotics, chloramphenicol and macroamphenicol, bind at different locations in the ribosome, disrupting different steps
of the protein synthesis process.
This has been predicted in studies of isolated ribosomes, but has never been observed in actual bacterial cells
.

"When we first saw drug molecules binding to ribosomes within cells, it was very exciting, but even more exciting, we found that the ribosome populations in antibiotic-treated cells were fundamentally reshaped
functionally, structurally, and spatially," Xue said.
”

The researchers observed that the interaction between the ribosome and other complexes within the cell changed under the action of the drug, suggesting that the antibiotic's effect may extend far beyond the specific complex to which it binds
.
"On the one hand, this can help understand the off-target effects of antibiotics, and it may also help design combinations of antibiotics to improve their efficiency," Mahamid said
.

Mahamid's group continues to harness the power of cryogenic ET to study basic biological processes
.
"What we were able to do for this extremely simple model system is in principle applicable to more complex models," Mahamid said
.
"For example, in our team, we study interactions between viruses and their human cell hosts, the tissue and ribosome function of human pluripotent stem cells, and even the large multicellular 3D organoids
that we culture with our collaborators and cells that we have directly extracted from cancer patients.
"

Visualizing translation dynamics at atomic detail inside a bacterial cell