A group of EMBL scientists is now using this unique organism to interrogate fundamental processes that underlie the biology of a living cell. Spearheaded by the research group of Julia Mahamid<\/a>, and enabled by collaborations with multiple groups within and outside EMBL, the researchers are attempting to ‘see’, at unprecedented resolution, the mechanisms of life inside one of the smallest living cells.\u00a0\u00a0<\/p>\n\n\n\n Researchers from EMBL\u2019s Structural and Computational Biology unit established Mycoplasma pneumoniae <\/em>as a model organism in the early 2000s. Teams led by Peer Bork<\/a>, Luis Serrano<\/a>, and Anne-Claude Gavin<\/a> sequenced and annotated its genome, which was followed by the publication of three seminal papers delineating its metabolomics, proteomics, and transcriptomics, respectively.\u00a0<\/p>\n\n\n\n Two decades earlier, Jacques Dubochet<\/a>, another EMBL researcher, had developed a way to prepare and image biological samples using cryo-electron microscopy (cryo-EM), work that won him the Nobel Prize in 2017. Relying on a method of freezing biological samples extremely quickly to prevent the formation of ice crystals, cryo-EM allows researchers to observe the structure of complex biomolecules at atomic resolution.\u00a0<\/p>\n\n\n\n Soon afterwards, researchers developed the technique of cryo-electron tomography (cryo-ET), another big leap in harnessing the power of electron microscopy. While cryo-EM allows scientists to observe the structure of biological molecules, it usually requires samples of carefully isolated molecules taken out of their cellular context. However, cryo-ET allows scientists to take snapshots of intact cells, along with all their internal components, which can later be reconstructed in 3D. <\/p>\n\n\n\n Given Mycoplasma\u2019s <\/em>small size, it is possible to obtain a series of images to combine into a clear picture of the entire cell and all its constituents. When Mahamid started her group at EMBL in 2017, she decided to harness the growing power and resolution of advanced cryo-ET technologies to study cellular mechanisms in action inside this model organism.<\/p>\n\n\n\n \u201cPersonally, I’m amazed by the ability to do structural biology inside cells,\u201d said Joe Dobbs, one of the PhD students in the Mahamid group. \u201cCryo-EM is well-known for its ability to provide insight into the structures of macromolecular complexes in purified samples, but actually seeing the insides of cells, and how molecules interact with each other in context, is incredible.\u201d<\/p>\n\n\n\n The study has attracted a diverse group of scientists and engineers. Vastly differing in areas of interest and expertise, they are united by their belief in the potential for M. pneumoniae <\/em>to serve as a window into the life of a cell. <\/p>\n\n\n\n For example, Judith Zaugg<\/a> started her lab at EMBL in 2014 and is interested in understanding the molecular basis of complex genetic traits and diseases, for which she usually works with human genomics data. With Mycoplasma, <\/em>she became fascinated by the idea of observing gene expression in action within a living cell. In the cryo-ET images that the Mahamid group grabs, DNA can be observed as filamentous structures and the RNA polymerase enzymes seen attached to them, and Zaugg believes that one day this might be used to figure out which regions of DNA or genes are active \u2013 or being \u2018transcribed\u2019 \u2013 at a given point in time.\u00a0<\/p>\n\n\n\n \u201cIt is very exciting to \u2018see\u2019 transcription in progress,\u201d said Zaugg. \u201cWith this model, we can perhaps one day address very fundamental biological questions like \u2013 how does the cell reorganise its DNA upon receiving a certain stimulus? While we know (based on genomics data) that genes are differentially expressed based on stimuli, we still don’t know how the cell rearranges its internal structure and genome to achieve this.\u201d <\/p>\n\n\n\n With future advances in correlative electron and fluorescence microscopy, Zaugg hopes that answering questions like these might become possible. In turn, her team brings to the project computational expertise to mine the data, which otherwise could have taken months or even years to process.<\/p>\n\n\n\n Peer Bork<\/a>, who co-initiated and coordinated the initial proteomic, transcriptomic, and metabolomic characterisation of M. pneumoniae <\/em>in the 2000s, is interested not only in the infectious disease aspects of this model system but also in its potential for understanding molecular networks. His group has aided the study with computational expertise as well as by connecting global bioinformatic data.\u00a0<\/p>\n\n\n\n \u201dThis project leverages some of EMBL\u2019s key strengths \u2013 collaborating and coordinating across a diversity of disciplines to answer fundamental biological questions that cannot be addressed otherwise,\u201d said Bork. <\/p>\n\n\n\n Maria Zimmermann-Kogadeeva<\/a>, another EMBL group leader, started collaborating with Mahamid while still a postdoc in the Bork group. Her computational insights played a key role in a recent project that studied the machinery of protein synthesis in action inside the cell. She is also involved in a project focused on studying previously uncharacterised membrane proteins that can be observed in cryo-ET images of Mycoplasma.\u00a0<\/em><\/p>\n\n\n\n \u201c<\/em>The cryo-ET technology allows us to look deep inside the cell, and understand how intracellular processes change under different conditions,\u201d said Zimmermann-Kogadeeva. \u201cThe data obtained is of very high quality and can be combined with other molecular datasets to ask interesting questions.\u201d She and Bork are both optimistic about the potential of investigating cellular metabolomics within Mycoplasma<\/em> in the future. <\/p>\n\n\n\n \u201cOur goal is to connect biology across scales,\u201d said Mahamid. \u201cMycoplasma <\/em>gives us a way to study biological systems all the way across from the nanometre range of molecules to the micrometre range of entire cells.\u201d <\/p>\n\n\n\n While cryo-ET allows scientists to look at the whole cell, it doesn\u2019t make it easy to identify what<\/em> they are seeing. Unlike with fluorescence microscopy, it isn’t easy to tag or label specific objects inside the cell and distinguish them from their neighbours in cryo-ET. As a result, scientists are effectively left with an incomplete map, most of which has no helpful labels. <\/p>\n\n\n\n Soheil Mojiri, a postdoctoral fellow in the Ries group<\/a>, is building a microscope to solve this problem. An engineer by training, Mojiri is fascinated by the possibility of exploiting optical physics to address challenging biological questions. His goal is to fruitfully marry cryo-ET and super-resolution optical microscopy at low temperatures.\u00a0<\/p>\n\n\n\n \u201cCryo-ET allows us to look at molecular architecture and molecules in context, but we’re limited by what we can identify in the extremely noisy, crowded, and heterogeneous cellular environment. So while we can just about make out big things, such as ribosomes – the cell’s protein production machines, smaller molecules and complexes can escape our notice unless we have prior information to use,\u201d said Mojiri. <\/p>\n\n\n\n Mojiri is developing a prototype microscope that would allow scientists to take a frozen cell sample, visualise individual proteins or complexes with fluorescent tags that are genetically introduced into live cells, and later subject the same sample to cryo-ET. Comparing the images obtained by the two methods would let researchers leverage electron microscopy\u2019s structural resolution as well as fluorescence microscopy\u2019s specificity. <\/p>\n\n\n\n Not all technology development for this project is on the hardware side, however. The researchers have generated a huge volume of data, which presents challenges in analysing it to draw new insights. This is where Anna Kreshuk<\/a>, with her expertise in machine-learning-based image analysis, steps in. Kreshuk and her team create innovative AI-based methods that find meaningful information from biological images, often gigabytes in size and containing a lot of background information that may not be relevant.\u00a0<\/p>\n\n\n\n Ricardo Sanchez, an ARISE fellow<\/a> at EMBL Heidelberg, has been working on finding ways around this challenge. \u201cWith cryo-ET, one of the biggest problems is reducing the signal-to-noise ratio and identifying structures of interest among all the cellular background,\u201d he said. \u201cMy goal is to solve this problem with the least amount of computational resources.\u201d<\/p>\n\n\n\n \u201cWhat fascinates me is the completeness of this system,\u201d said Kreshuk. \u201cIt’s the whole living thing. Everything that it needs to be an independent (and pretty dangerous) living organism is visible in that one image.\u201d<\/p>\n\n\n\n The project is already yielding dividends when it comes to understanding fundamental biological processes that make life possible. In a recently published paper, Liang Xue from the Mahamid group led a study that allowed the team to visualise the process of translation \u2013 the synthesis of new proteins \u2013 inside the cell at atomic detail. The structures they focused on is probably one of the easiest to identify in a cryo-ET image \u2013 ribosomes. Ribosomes are some of the most ancient molecular machines present in all living organisms and essential for protein synthesis. <\/p>\n\n\n\n \u201cRibosomes not only play an essential role in genetic information flow but also serve as platforms to monitor cellular states, e.g. cell stress response,\u201d said Xue. \u201cInside living cells, ribosomes function as highly interconnected networks of molecular machines.\u201d <\/p>\n\n\n\n Using high-quality cryo-ET Mycoplasma <\/em>data, <\/em>the researchers were not only able to observe the dynamic structural changes that took place in ribosomes as they proceeded through the protein synthesis cycle, but they could also observe what happens to these processes when antibiotics perturb cells. Since the translation machinery is quite similar in structure and function throughout the tree of life, the study can be extrapolated to other prokaryotic or eukaryotic species that are more challenging to image with cryo-ET. <\/p>\n\n\n\n \u201cI hope my work at EMBL establishes a framework<\/a> to do high-resolution<\/a> structural biology inside the cell,\u201d said Xue. \u201cRibosomes and Mycoplasma<\/em> are just the beginning. With more and more molecular machines resolved inside cells, we are heading toward the ultimate goal of building an atomic cell model.\u201d<\/p>\n\n\n\n The researchers are also using Mycoplasma <\/em>to understand the role of unknown membrane proteins that can be observed in cryo-ET images In these studies, Jan Kosinski<\/a>\u2019s and Christian L\u00f6w<\/a>\u2019s groups at EMBL Hamburg and Nassos Typas<\/a>\u2019s group at EMBL Heidelberg are also involved. A tool they have turned to often in these investigations is AlphaFold<\/a>, the AI algorithm that predicts protein structures, whose developers won the 2023 Breakthrough Prize in life sciences.\u00a0<\/p>\n\n\n\n The project also involves collaborations with scientists outside EMBL and in EMBL member states, including the groups of J\u00f6rg St\u00fclke<\/a>, Georg-August-Universit\u00e4t G\u00f6ttingen, Patrick Cramer<\/a>, Max Planck Institute for Multidisciplinary Sciences, Juri Rappsilber<\/a>, Institut f\u00fcr Biotechnologie, Technische Universit\u00e4t Berlin, and Luis Serrano<\/a>, Centre for Genomic Regulation, Barcelona.<\/p>\n\n\n\n While research in the last few decades has helped to rapidly identify and annotate many species\u2019 genomes, our knowledge of the function and precise roles of most genes and proteins remains incomplete, even for an organism as simple as Mycoplasma<\/em>. Projects like these are a key step towards bridging this knowledge gap. EMBL\u2019s new programme \u2018Molecules to Ecosystems\u2019<\/a> aims to study living organisms in the context of their environment. In doing so, scientists will doubtless encounter many more new molecules and biological processes, which this project paves the way towards studying and understanding in detail. It also exemplifies the collaborative approaches at the heart of this new programme.<\/p>\n\n\n\n \u201cI think it is a pretty long and hard way from imaging Mycoplasma<\/em> to the data interpretation. We need diverse expertise and knowledge to pave this way,\u201d said Mojiri. \u201cIn particular, we require biologists, physicists, and data scientists.\u201d The collaborative atmosphere at EMBL has made such a coming together of talents and knowledge possible. The team gathers in a monthly meeting dedicated to the Mycoplasma<\/em> project to exchange ideas, discuss the progress of experiments, and troubleshoot challenges.<\/p>\n\n\n\n Researchers have observed the inner workings of an unusual bacteria at an unprecedented level of detail.<\/p>\n","protected":false},"author":124,"featured_media":53308,"parent":0,"menu_order":0,"template":"","tags":[],"class_list":["post-53292","embletc","type-embletc","status-publish","has-post-thumbnail","hentry"],"acf":{"featured":true,"show_featured_image":false,"field_target_display":"embl","field_article_language":{"value":"english","label":"English"},"article_intro":" Researchers have observed the inner workings of an unusual bacteria at an unprecedented level of detail<\/p>\n","related_links":[{"link_description":"Seeing deeper inside cells\r\n","link_url":"https:\/\/www.embl.org\/news\/science\/seeing-deeper-inside-cells\/"},{"link_description":"Seeing antibiotics in action inside a pathogenic bacterium\r\n","link_url":"https:\/\/www.embl.org\/news\/science\/seeing-antibiotics-in-action-inside-a-pathogenic-bacterium\/"},{"link_description":"Mahamid Group","link_url":"https:\/\/www.embl.org\/groups\/mahamid\/"}],"source_article":false,"in_this_article":false,"press_contact":"None","article_translations":false,"languages":"","embletc_issue":[{"ID":53290,"post_author":"124","post_date":"2022-11-16 12:00:00","post_date_gmt":"2022-11-16 11:00:00","post_content":"","post_title":"Issue 99","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"issue-99","to_ping":"","pinged":"","post_modified":"2022-11-17 11:34:48","post_modified_gmt":"2022-11-17 10:34:48","post_content_filtered":"","post_parent":0,"guid":"https:\/\/www.embl.org\/news\/?post_type=embletc-issue&p=53290","menu_order":0,"post_type":"embletc-issue","post_mime_type":"","comment_count":"0","filter":"raw"}],"embletc_in_this_issue":[{"ID":53298,"post_author":"96","post_date":"2022-11-16 12:00:00","post_date_gmt":"2022-11-16 11:00:00","post_content":"\n Sunlight delivers precious energy that organisms capture using specialised molecular \u2018solar panels\u2019. Only two kinds of molecules can achieve this: chlorophyll-based proteins, which enable photosynthesis, and rhodopsins. Besides capturing solar energy, rhodopsins are also present in the retina and enable us to see.<\/p>\n\n\n\n In contrast to green chlorophyll-based proteins, which dominate on land, rhodopsins are mostly purple and found in aquatic microorganisms, especially ocean-living bacteria, archaea, algae, and even some giant viruses. <\/p>\n\n\n\n Kirill Kovalev, an EIPOD<\/a> postdoc in the Schneider Group<\/a> at EMBL Hamburg and the Bateman Group<\/a> at EMBL-EBI, is fascinated by microbial rhodopsins. Trained as a physicist, he uses cutting-edge structural biology techniques to create molecular stop-motion visualisations showing how rhodopsins change their molecular structure to capture solar energy. With this knowledge, he designs new, more powerful rhodopsins that neuroscientists could apply as tools to control neuronal activity.<\/p>\n\n\n\n Why are rhodopsins and chlorophylls so colourful? The colour that we see is the light that these molecules reflect while absorbing all other wavelengths. For example, rhodopsins capture green and blue light and reflect purple wavelengths. Different types of rhodopsins reflect slightly different ranges of wavelengths, which gives them different hues of purple, violet, pink, and orange. Which wavelengths are absorbed or reflected is determined by the molecule\u2019s structure. Rhodopsins have a simpler structure than chlorophylls and are believed to be evolutionarily older.<\/p>\n\n\n\n Rhodopsins\u2019 ability to absorb specific light wavelengths not only gives them their pretty colours but also makes them scientifically interesting. In particular, one group of rhodopsins found in algae, called channelrhodopsins, is studied for its unique ability to trigger electrical activity in cells when exposed to blue light. <\/p>\n\n\n\n Scientists have adapted channelrhodopsins for human and animal cells, such that they can be used like an on- and off-switch for cellular activity. This technique, called \u2018optogenetics\u2019, was a game-changer for neuroscientists because it enables them to precisely and quickly stimulate selected neurons in the brain with just a flash of light. <\/p>\n\n\n\n Currently, scientists have only a few channelrhodopsins to pick from, which makes certain experiments difficult or even impossible. To address this, Kovalev is on the search for new, more powerful rhodopsins that could also be activated with other colours of light.<\/p>\n\n\n\n Using X-rays, he compares different rhodopsins found in bacteria, often in exotic places, such as salty lakes or glaciers. This lets him understand how tiny differences in the molecular structure determine protein properties and function, for instance, which colours a particular rhodopsin is sensitive to. <\/p>\n\n\n\n \u201cOften, we are comparing almost identical rhodopsin variants,\u201d said Kovalev. \u201cA difference of just one atom is enough to change the molecule\u2019s properties. That\u2019s why we need to analyse them at atomic resolution.\u201d<\/p>\n\n\n\n One advantage of these comparisons is that they let Kovalev predict which modifications would make rhodopsins responsive to new colours. Then, he and his collaborators can create these rhodopsins in the laboratory and test them. <\/p>\n\n\n\n What happens when a rhodopsin absorbs light? First, it gives rise to a complicated series of changes in the structure of the rhodopsin molecule. To visualise this process in detail, Kovalev uses a technique called time-resolved X-ray crystallography at EMBL Hamburg\u2019s beamline P14. This experimental facility is adapted for particularly demanding crystallography experiments. X-ray crystallography enables Kovalev to take snapshots of a rhodopsin at specific timepoints after absorbing light and combine them into a stop-motion movie. <\/p>\n\n\n\n \u201cStudying a molecule that undergoes so many changes within just milliseconds is extremely challenging. Besides, rhodopsins are membrane proteins. Such proteins are notoriously difficult to crystallise, which makes them even trickier to work with,\u201d said Kovalev. \u201cBut I can do this at EMBL Hamburg\u2019s beamline P14 at PETRA III, which is one of the best in the world for this type of experiments.\u201d <\/p>\n\n\n\n Kovalev was one of the first at EMBL Hamburg to attempt a time-resolved crystallography experiment on a membrane protein. Before he could start experiments, the Schneider Group worked together, each member contributing different expertise, to make the beamline setup suitable for Kovalev\u2019s project. Besides adjustments for membrane proteins, they installed a system to emit a flash of light to activate the rhodopsins, coordinated with the X-ray beam that would take a snapshot precisely at a selected timepoint milliseconds later. <\/p>\n\n\n\n In addition to the EMBL beamline P14, Kirill also performs experiments at the European XFEL<\/a>, which allows for time resolution in the range of femtoseconds (one quadrillionth of a second). <\/p>\n\n\n\n \u201cThis combination of having a synchrotron and XFEL in one place is very helpful. Besides Hamburg, only Japan and Switzerland have a combo like this,\u201d said Kovalev.<\/p>\n\n\n\n In the coming years, time-resolved experiments at EMBL Hamburg, such as this, will be further improved upon to observe how proteins work with even better time resolution. This will be possible with the planned upgrade of the synchrotron PETRA III to PETRA IV<\/a>. <\/p>\n\n\n\n Kovalev, a physicist by training, first started to work on rhodopsins during his bachelor\u2019s studies. <\/p>\n\n\n\n \u201cWhat got me interested in biology was the opportunity to apply physics methods to solve unsolved problems. I like the thrill of being the first one to see how the molecule moves and understand how it works. I also like that my work can potentially help others in research and medicine,\u201d he said.<\/p>\n\n\n\nFrom pathogen to model<\/strong><\/h2>\n\n\n\n
Connecting biology across scales<\/strong><\/h2>\n\n\n\n
New tools for new insights<\/strong><\/h2>\n\n\n\n
![\"Two](\"https:\/\/www.embl.org\/news\/wp-content\/uploads\/2022\/11\/EMBL-etc_005_retouched-1024x683.jpg\")
![\"Three](\"https:\/\/www.embl.org\/news\/wp-content\/uploads\/2022\/11\/CZI_awards-wp.jpg\")
Putting together a cellular picture<\/strong><\/h2>\n\n\n\n
Collaborating to make progress<\/strong><\/h2>\n\n\n\n
![\"A](\"https:\/\/www.embl.org\/news\/wp-content\/uploads\/2022\/11\/EMBL-etc_010-1024x683.jpg\")
\u201cMycoplasma <\/em>has turned out to be a powerful model for studying the dynamics of molecular machines and understanding intracellular cross-talk,\u201d said Mahamid. \u201cThe collaborative approach was absolutely crucial to this. To realise the massive potential of cryo-ET and the Mycoplasma <\/em>model, we will need to continue as we have started, together and moving forward.\u201d<\/p>\n","protected":false},"excerpt":{"rendered":"The many colours of molecular solar panels<\/strong><\/h2>\n\n\n\n
![\"Shoreline](\"https:\/\/www.embl.org\/news\/wp-content\/uploads\/2022\/11\/AdobeStock_456172831-1-1024x673.jpeg\")
Credit: Adobe Stock.<\/figcaption><\/figure>\n\n\n\nSwitching cells on and off with light<\/strong><\/h2>\n\n\n\n
Expanding the rhodopsin colour palette<\/strong><\/h2>\n\n\n\n
![\"Over](\"https:\/\/www.embl.org\/news\/wp-content\/uploads\/2022\/11\/Hamburg_Profile_39-Kirill_Kovalev-1024x683.jpg\")
Rhodopsins in stop-motion<\/strong><\/h2>\n\n\n\n
![\"Closeup](\"https:\/\/www.embl.org\/news\/wp-content\/uploads\/2022\/11\/Hamburg_Profile_34-1024x683.jpg\")
Using light to hear better<\/strong><\/h2>\n\n\n\n
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