An advance in cryo-electron microscopy instrumentation will enable the mechanisms of antibiotic resistance due to bacterial ribosome mutations to be understood more rapidly.
By refining a technique known as cryo-electron microscopy, researchers from Imperial College London and CNRS-Inserm-Strasbourg University have determined how the enzyme RF3 helps prepare the protein-making factory for its next task following disconnection of the newly formed protein strand.
The team's success in capturing the protein-making factory, or ribosome, in action using cryo-electron microscopy will help scientists to begin deciphering the molecular detail of how many antibiotics interfere with the final steps of protein synthesis - an area not currently targeted in antibiotics research.
Professor Marin van Heel of Imperial's Department of Biological Sciences and senior author of the study says:
"Many antibiotics kill bacteria by interfering with their protein-making factories, or ribosomes. But bacteria can often become resistant by mutating their ribosome machinery. Observing ribosomes in action helps us understand which areas of the protein complex evolve such resistance quickly. This information could then be used to develop new antibiotics that target the more stable regions.
"We've used cryo-electron microscopy in a similar way to time lapse footage. It has allowed us to visualise how one cog in a cell's protein manufacturing plant operates. By refining the technique even further we hope to be able to visualise the molecular interactions on an atomic scale. This kind of knowledge has applications across the board when you are trying to work out key factors in diagnosis, treatment or cause of many diseases."
Professor van Heel pioneered cryo-electron microscopy 10 years ago. Since then it has become an essential part of many structural biologists' toolkit. It overcomes the problem of weak image contrast in electron microscopy and avoids the difficult and time-consuming process of growing crystals that can be analysed using X-ray diffraction.
As professor van Heel points out, this technique is applicable to the study of the shape and action of many other types of molecules in cells.
Rapid freezing provides a snapshot of what ribosomes were doing at that moment of time. Also, the freezing prevents higher electron doses from changing the shape of the ribosomes and hence a larger electron dose can be used to get a clearer picture. This is analogous to how a bright camera flashbulb provides more light to get a better picture.
Electron microscopy images are created by firing electrons at the sample but this process rapidly damages the biological material. To overcome this degradation problem researchers use a low dose of radiation, which leads to extremely poor image quality. "It's like trying to see in the dark," says Professor van Heel.
"Cryo-electron microscopy uses a sample in solution which is rapidly frozen by plunging it into liquid ethane and maintaining it at the same temperature as liquid nitrogen," he explains.
"This maintains the 3D structure of the molecule and gives you instant access to the cellular process of interest. Also, the effect of freezing the sample before electron microscopy reduces radiation damage. This makes it possible to apply a higher electron dose, which gives a clearer image."
The goal is to create a movie of the molecular level changes that happen to ribosomes as they perform protein synthesis.
"After the X-ray structure of the ribosome became available a few years ago, one might think we already know all there is to know about protein synthesis," explains Professor van Heel.
"But we've still got so much to learn about the precisely synchronised series of steps that occurs. Researchers only became aware of the existence of ribosomes 50 years ago but they've a mystery since the creation of life 3.5 billion years ago. By improving the high resolution images we can create using cryo-electron microscopy our long term goal is to create a movie of protein synthesis on an atomic scale."
Advances in instrumentation speed up the rate of advances in basic biological science, biomedical research, and biotechnology by providng scientists with better tools for watching and manipulating biological systems. As a result each year witnesses a faster rate of discovery than the year previous. When people ask when various diseases will be cured what they ought to ask is when will the tools biologists have to use become advanced enough to enable biologists to figure out the causes and to develop effective treatments? While that is perhaps a more precise question it is still very difficult to answer.
|Share |||Randall Parker, 2004 February 27 09:20 AM Biotech Advance Rates|