Tag: Cryo EM

What role does Elongator play in brain development?

What role does Elongator play in brain development?

What role does a tRNA modification complex, called Elongator, play in brain development?

SOLARIS Centre users from the Malopolska Centre of Biotechnology (of the Jagiellonian University, together with Australian, Turkish and Canadian colleagues, have found a link between defects in the cellular protein production machinery and neurodevelopmental disorders (NDDs), characterized by an inability to reach cognitive and motor milestones. Key studies in this publication were conducted using Cryo-EM microscopes located at our center.

The speed rate of protein synthesis is crucial to the integrity of the proteome

Scientists showed how genetic mutations in patients affect the Elongator activity and lead to severe clinical symptoms. The study provided the first clinical evidence for missense mutations in the Elongator accessory subcomplex ELP456 to cause neurodevelopmental disorders. Genome-wide analysis allowed identification of pathogenic variants in patients with severe clinical presentation of NDDs. Further modelling of the patient-derived mutations in mice resembled the complex neurodevelopmental phenotype and revealed neuron-specific consequences of the found genetic mutations.

„We report patient-derived substitutions in the accessory ELP456 subcomplex to affect different types of neurons than previously known mutations in the catalytic core of the complex” – explains Dr. hab. Sebastian Glatt, the senior author and head of the Max Planck Research Group, that carried out the experimental work in Krakow. This provides a novel concept in the field that depletion of specific tRNA modifications in patient cells may induce specific changes in the cellular proteomes.

Read more on the SOLARIS website

Arranging gold nanoparticles precisely in three dimensions

Arranging gold nanoparticles precisely in three dimensions

Metal nanoparticles have a wide variety of applications many of which stem from the fact that extremely small particles a few nanometres to  10’s of nanometres in diameter can have very different properties from those of the same material at a larger scale (a nanometre is just a billionth of a metre). Such particles are used as catalysts, coloring agents and can even  make antibacterial coatings. Some effects are due to the pattern of the particles and the spacing between them, but these are very difficult to control and particles are typically used in solution where they randomly move around like motes of dust in the air.   

In the current work, scientists based at the Bionanoscience and Biochemistry Laboratory at the Malopolska Centre of Biotechnology (MCB), Jagiellonian University showed that an artificial protein structure, a hollow sphere called a TRAP-cage, was able to act as a scaffold and provide regular-spaced points of attachment for small gold nanoparticles. “TRAP-cage is itself tiny, but at around 15 nm in diameter is still big enough to attach multiple  gold nanoparticles” explained Jonathan Heddle the head of the lab, “The protein cage is made of 12 rings, so overall it looks a little like a 12-sided dice – a dodecahedron.”  The researchers showed that there are spaces equivalent to the corners of the dodecahedron that offer just the right environment to snugly fit the gold nanoparticles inside. As a result, instead of randomly floating around, the particles appear to be constrained into a fixed three-dimensional pattern. It is hoped that the ability to arrange metal nanoparticles in this way may be developed further to produce new materials with useful properties.

Read more on the SOLARIS website

Image: The structure of the protein cage (purple) with three of the embedded gold nanoparticles highlighted (yellow) 

Credit: Jonathan Heddle

Science Advances cover dedicated to research results on Cryo-EM

Science Advances cover dedicated to research results on Cryo-EM

The research carried out at NCPS SOLARIS with the use of electron cryomicroscopy and at the Malopolska Biotechnology Centre, and at the British National Electron Bioimaging Center eBIC (Diamond Light Source) allowed to solve the structure of the protein responsible for introducing compounds necessary for the life of bacterial cells. The exceptional importance of the research was honored with a dedicated, unique image by Alina Kurokhtina published on the cover of Science Advances!

Bacterial species are under continuous warfare with each other for access to nutrients. To gain an advantage in this struggle, they produce antibacterial compounds that target and kill their competitors. Different species of bacteria, including ones that live inside us, can battle each other for scarce resources using a variety of tactics. Now, researchers from the laboratories of Prof Jonathan Heddle from Malopolska Centre of Biotechnology, Jagiellonian University, Krakow and Dr Konstantinos Beis at Research Complex at Harwell /Imperial College, London, have uncovered the mechanism of one such tactic in work that may eventually lead to the development of new antibacterials.

Read more on the SOLARIS website

Image: A view of the determined SbmA structure in gold

Credit: Alina Kurokhtina

Retrovirus research using Cryo-EM

Retrovirus research using Cryo-EM

Reverse transcription involves the conversion of single-stranded RNA to double-stranded DNA. This is a key step in the replication of retroviruses, catalyzed by the enzyme reverse transcriptase. Retroviruses are divided into two subfamilies, one of which, Spumaretrovirinae, has a different proliferation cycle and a different reverse transcriptase domain structure. The presented studies provide the first structural description of the nucleic acid binding by viral reverse transferase, demonstrating its ability to change the oligomeric state depending on the type of bound nucleic acid.

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA, a crucial process for the replication of retroviruses. Foamy viruses (FV) possess a unique RT which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. The authors report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. Moreover, the describtion of a structure of MFV PR-RT with RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer has been presented. Cryo-electron microscopy reconstruction of full-length MFV PR-RT – dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. 

Read more on the SOLARIS website

Image: Model of FV

New Data sheds light on genesis of our body’s powerhouses

New Data sheds light on genesis of our body’s powerhouses

The mitochondria and its protein making “plants” – mitoribosomes

Scientists uncover for the first time how the body’s energy makers are made using Cryo-Electron Microscopy (cryo-EM) at eBIC within Diamond.

A new paper, published in Science on the 19th February, by an international team of researchers reports an insight into ‘the molecular mechanism of membrane-tethered protein synthesis in mitochondria’. This is a fundamental understanding of how the human mitoribosome functions and could explain how it is affected by mutations and deregulation that lead to disorders such as deafness and diseases including cancer development. 

Mitochondria are intracellular organelles which serve as tiny but potent powerhouses in our body. They use oxygen which we inhale and derivatives from food we eat to produce more than 90% of our energy, and therefore effectively support our life. Mitochondria are particularly important in high-energy demanding organs such as heart, liver, muscles and brain. For example, almost 40% of each heart muscle cell is made up of mitochondria.

Read more on the Diamond website

Image: The mitoribosome is attached to its membrane adaptor as it synthesises a bioenergetic protein (glow yellow).

Credit: Dan W. Nowakowski and Alexey Amunts

Blood disorder mechanism discovered

Blood disorder mechanism discovered

G6PD deficiency affects about 400M people worldwide and can pose serious health risks. Uncovering the causes of the most severe cases could finally lead to treatments.

With a name like glucose-6-phosphate dehydrogenase deficiency, one would think it is a rare and obscure medical condition, but that’s far from the truth. Roughly 400 million people worldwide live with potential of blood disorders due to the enzyme deficiency. While some people are asymptomatic, others suffer from jaundice, ruptured red blood cells and, in the worst cases, kidney failure. 

Now, a team led by researchers at the Department of Energy’s SLAC National Accelerator Laboratory has uncovered the elusive mechanism behind the most severe cases of the disease: a broken chain of amino acids that warps the shape of the condition’s namesake protein, G6PD. The team, led by SLAC Professor Soichi Wakatsuki, report their findings January 18th in Proceedings of the National Academy of Sciences. 

Read more on the SLAC website

Image: The G6PD enzyme plays a crucial role in red blood cells, removing molecules such as hydrogen peroxide from the body. In some cases, mutations can bend the molecule awkwardly, interfering with G6PD’s function. In the worst cases, the mutations lead red blood cells to rupture.

Credit: Mio Wakatsuki, from protein images by Naoki Horikoshi/SLAC National Accelerator Laboratory

Science Begins at Brookhaven Lab’s New Cryo-EM Research Facility

Science Begins at Brookhaven Lab’s New Cryo-EM Research Facility

Brookhaven Lab’s Laboratory for BioMolecular Structure is now open for experiments with visiting researchers using two NY State-funded cryo-electron microscopes.

UPTON, NY—On January 8, 2021, the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory welcomed the first virtually visiting researchers to the Laboratory for BioMolecular Structure (LBMS), a new cryo-electron microscopy facility. DOE’s Office of Science funds operations at this new national resource, while funding for the initial construction and instrument costs was provided by NY State. This state-of-the-art research center for life sciences imaging offers researchers access to advanced cryo-electron microscopes (cryo-EM) for studying complex proteins as well as the architecture of cells and tissues.

Many modern advances in biology, medicine, and biotechnology were made possible by researchers learning how biological structures such as proteins, tissues, and cells interact with each other. But to truly reveal their function as well as the role they play in diseases, scientists need to visualize these structures at the atomic level. By creating high-resolution images of biological structure using cryo-EMs, researchers can accelerate advances in many fields including drug discovery, biofuel development, and medical treatments.

Read more on the BNL website

Image: Brookhaven Lab Scientist Guobin Hu loaded the samples sent from researchers at Baylor College of Medicine into the new cryo-EM at LBMS.

Diamond helps uncover how an untreatable cancer-causing virus affects immune cells

Diamond helps uncover how an untreatable cancer-causing virus affects immune cells

Scientists have found that human T-cell lymphotropic virus, type 1 (HTLV-1) hijacks cellular machinery to establish an infection.  

Research was undertaken using cutting-edge visualisation techniques such as X-ray crystallography, which was undertaken at Diamond, and single-particle cryo-electron microscopy (cryo-EM).  

HTLV-1 is a virus that affects T cells, a type of white blood cell which plays a crucial role in our immune system. Currently, between five and 20 million people worldwide are infected by HTLV-1 and no cure or treatment is available. While most people infected with the virus do not experience symptoms, around two to five per cent will go on to develop adult T-cell leukaemia (ATL).  

New research, led by a team from Imperial College London and the Francis Crick Institute, shows in atomic detail how HTLV-1 infects immune cells. By providing a more nuanced understanding of how the virus establishes infection in the body, the research will help to support the development of new, targeted therapies. 

Read more on the Diamond Light Source website

Image: Scanning electron micrograph of a human T lymphocyte (also called a T cell) from the immune system of a healthy donor. Credit: NIAID

Cryo-electron microscopy for industry coming soon to SOLARIS

Cryo-electron microscopy for industry coming soon to SOLARIS

The SOLARIS Centre and the Malopolska Centre of Biotechnology (Jagiellonian University) won a two-stage competition for the purchase of an electron microscopy for industrial research.

Funding was awarded by the National Information Processing Institute (OPI PIB) as part of the EU’s Smart Growth Operational Programme.

“We have been trying to purchase a microscope because Polish companies keep asking us about the possibility to carry out measurements using the Cryo-EM technique” – says Michał Młynarczyk, Finance and Administration Deputy Director at SOLARIS. “We expect that the total time allocated for the commercial study will be at least 40% of operational time. The remaining time will be available for academic researchers” –  continues the director.

“We are keen to enable Polish companies to access this exciting new technology, which is developing very fast and is currently becoming the most important one used in structural biology. The achievable results facilitate to understand the cellular mechanisms behind human diseases, the design of new drugs, the optimization of existing drug molecules. The technique is also successfully applied in nanotechnology and other fields” – adds Sebastian Glatt, Max Planck Research Group leader at the Malopolska Centre of Biotechnology – the main partner of SOLARIS in the implementation of this project.

>Read more on the SOLARIS website

First structure of a DNA crosslink repair ligase determined

First structure of a DNA crosslink repair ligase determined

Diamond’s Electron Bio-Imaging Facility (eBIC) has been used to generate the first 3D structure of the Fanconi anaemia (FA) core complex, a multi-subunit E3 ubiquitin ligase required for the repair of damaged DNA. The work, led by Dr Lori Passmore from the MRC Laboratory of Molecular Biology and a team of researchers, has been published today in Nature, and their research provides the molecular architecture of the FA core complex and new insights into how the complex functions.

The FA pathway senses and repairs DNA crosslinks that occur after exposure to chemicals including chemotherapeutic agents and alcohol, but also as a result of normal cellular metabolism. The megadalton FA core complex acts as an E3 ubiquitin ligase to initiate removal of these DNA crosslinks, helping to repair the damage caused. The research team used eBIC’s imaging facilities to make a major breakthrough in understanding the FA core complex by determining its structure using an integrative approach including cryo-electron microscopy and mass spectrometry.

Dr Peijun Zhang, Director of eBIC notes that:

Enabling cutting-edge research like this is exactly why we established eBIC, to provide scientists with state-of-the-art experimental equipment and expertise in the field of cryo-electron microscopy, for both single particle analysis and cryo-electron tomography. Determining the structure of the FA core complex for the first time is a fantastic achievement for the MRC research team.

>Read more on the Diamond Light Source website

Image: The FA core complex.
Credit: Phospho Biomedical Animation

Structural insights into tiny bacterial harpoons

Structural insights into tiny bacterial harpoons

Bacteria produce complex nano-harpoons on their cell surface. One of their functions is to harpoon and inject toxins into cells that are close by. Producing such a complex weapon requires lots of different moving components that scientists are still trying to understand. Researchers from the University of Sheffield have been using some of Diamond’s crystallography beamlines to understand a particularly enigmatic piece of this tiny puzzle. The team led by David Rice and Mark Thomas worked on a protein component of the harpoon called TssA which they already knew was an integral piece of the machinery. However, unlike the other components of the harpoon, there are distinct variants of the TssA protein that contain radically different amino acid sequences at one end of the protein. The team showed that the structures of the variable region of two different TssA subunits were completely unrelated and they could assemble into distinctly different multisubunit complexes in terms of their size and geometry. This begged the question as to how different bacteria could use this protein with different structures to produce a harpoon with the same function across all species. They found that despite these differences, there was a very specific conserved region at the other end of the protein. They hypothesise that the conserved region is the part that does the work and helps the harpoon to function whereas the variable region acts as a scaffold. They used I02, I03 and I24 in their study and plan to do follow up work using X-ray crystallography and Cryo-EM such as those at the eBIC centre at Diamond. The research was published in Nature Communications.

>Read more on the Diamond Light Source website

Image: Macromolecular Crystallography (MX) at Diamond reveals the shape and arrangement of biological molecules at atomic resolution, knowledge of which provides a highly accurate insight into function. 

Construction starts on new Cryo-EM center

Construction starts on new Cryo-EM center

Called the Laboratory of BioMolecular Structure, the new cryo-electron microscope center will offer world-leading imaging capabilities for life sciences research.

Today, the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory broke ground on the Laboratory of BioMolecular Structure (LBMS), a state-of-the-art research center for life science imaging. At the heart of the center will be two new NY-State-funded cryo-electron microscopes (cryo-EM) specialized for studying biomaterials, such as complex protein structures.

“Cryo-electron microscopy is a rapidly-advancing imaging technique that is posting impressive results on a weekly basis,” said LBMS Director Sean McSweeney. “The mission of LBMS is to advance the scientific understanding of key biological processes and fundamental molecular structures.”

“Throughout my career, I have worked hard to make our region of the State a high-tech hub, bringing together the talents and expertise of scientists and facilities across Long Island.  I am pleased to have played a part in the creation of the new cryo-EM center, which will add to the incredible facilities at Brookhaven National Lab and enable our scientific community to lead the way in world-class imaging research and discovery,” said NY State Senator Ken LaValle.

>Read more on the NSLS-II at BNL website

Image: New York State Senator Ken LaValle joined leaders of Empire State Development and Brookhaven Lab for the LBMS groundbreaking ceremony. Pictured from left to right are Jim Misewich (Associate Laboratory Director for Energy and Photon Sciences, Brookhaven Lab), Erik Johnson (NSLS-II Deputy for Construction), Sean McSweeney (LBMS Director and NSLS-II Structural Biology Program Manager), Robert Gordon (DOE-Brookhaven Site Office Manager), Ken LaValle, Cara Longworth (Regional Director, Empire State Development), Danah Alexander (Senior Project Manager, Empire State Development), and John Hill (NSLS-II Director).

Secrets of the deadly white-tail virus revealed

Secrets of the deadly white-tail virus revealed

The inner workings of a lethal giant freshwater prawn virus have been revealed by an international team of researchers using data gathered at Diamond Light Source. The results reveal a possible new class of virus and presents the prospect of tackling a disease that can devastate prawn farms around the world.

The detailed structure of a virus that can devastate valuable freshwater prawn fisheries has been revealed by an international team using image data collected in the Electron Bio-Imaging Centre (eBIC) based at Diamond Light Source. The researchers produced high-resolution images of virus like particles, VLP’s, composed of virus shell proteins which they compared with lower resolution images of the complete virus purified from prawn larvae. They found strong similarities between the two suggesting that the more detailed VLP images are a good representation of the intact virus. This research, exposing the inner workings of the MrNV, could make it easier to develop ways of combating the economically important disease, but also suggests that it belongs in a new, separate, group of nodaviruses.
The researchers used the rapidly developing technique of cryo-electron microscopy, cryoEM, which has the ability to produce very high-resolution images of frozen virus particles. Images so detailed that the positions of individual atoms could be inferred. Recent breakthroughs in this technique have transformed the study of relatively large biological complexes like viruses allowing researchers to determine their structures comparatively quickly. The data to produce the MrNV structure described here was captured in two days at the eBIC facility.

>Read more on the Diamond Light Source website

Image: 3D model of the MrNV
Credit: Dr David Bhella

First users on VMXm

First users on VMXm

First users from the University of Southampton investigated proteins involved in nutrient uptake of photosynthetic or cyanobacteria to understand how these phytoplankton thrive under scarce nutrient conditions.

The work has immense global significance for biofuels production and biotechnology. This beamline marks the completion of Diamond’s original Phase III funding on time and within budget.

First users have now been welcomed by Diamond Light Source, the UK’s national synchrotron light source on its new VMXm beamline. The Versatile Macromolecular Crystallography micro/nanofocus (VMXm) beamline becomes the 32nd operational beamline to open its doors to users, completing the portfolio of seven beamlines dedicated to macromolecular crystallography.
The unique VMXm beamline represents a significant landmark for Diamond. It is a specialist tuneable micro/nanofocus macromolecular crystallography (MX) beamline, with an X-ray beam size of less than 0.5 microns, allowing even the tiniest of samples to be analysed. Integrated into the ‘in vacuum’ sample environment is a scanning electron microscope, making VMXm a hybrid X-ray/cryoEM instrument for detecting and measuring data from nanocrystals. VMXm is aimed at research applications where the production of significant quantities of protein and crystals is difficult.

>Read more on the Diamond Light Source website

Image: Principal Beamline Scientist Dr Gwyndaf Evans with his team Dr Jose Trincao, Dr Anna Warren, Dr Emma Beale and Dr Adam Crawshaw. First users – Dr Ivo Tews from Biological Sciences at the University of Southampton and joint Diamond-Southampton PhD student Rachel Bolton investigating proteins involved in nutrient uptake of photosynthetic or cyanobacteria.

Funds for the latest generation of electron cryomicroscopy

Funds for the latest generation of electron cryomicroscopy

The Polish Ministry of Science and Higher Education handed over to SOLARIS the official decision to establish the National Cryo-EM Centre at the Polish partner facility, granting the requested financial support.

The successful application is the result of an agreement and cooperation of 17 leading scientific institutions in Poland in the area of structural biology. This very unique nation-wide consortium, led by Dr. Sebastian Glatt (the Malopolska Centre of Biotechnology, Jagiellonian University, Kraków) and Dr. hab. Marcin Nowotny (the International Institute of Molecular and Cell Biology, Warsaw), was not only key to bring this breakthrough research technique to Poland, but also exemplifies how scientists from around the country are able to work efficiently together for a greater common goal. This state-of-the-art microscope will allow its users to follow the progress of other international research centres and will transfer Polish and international scientists into the first class of structural biology.

The advances made in cryo-EM have revolutionized the field of structural biology over the last decade. The increased recognition of this technology has also culminated in the Chemistry Nobel Prize being awarded to its creators in 2017. The development of this technique has opened up new research horizons, which resulted in a long list of groundbreaking studies published in the most prestigious scientific journals. Foremost, the anticipated results are extremely relevant for a better understanding of the function of the human body, of the formation of human diseases and of processes like aging, and can lead to the development of new effective therapies. Structural biology has already contributed to a huge progress in the treatment of various human diseases, including cancer, Alzheimer’s disease and obesity. Last but not least, the presence of a high-end cryo-electron microscope at SOLARIS means that Krakow will attract national and international structural biologists.

>Read more on the SOLARIS website

Image: The image of mimivirus made with the use of a cryo-electron microscope.
Credit: Xiao C, Kuznetsov YG, Sun S, Hafenstein SL, Kostyuchenko VA, et al. (2009) [CC BY 2.5]

The ESRF CryoEM excels in its first year

The ESRF CryoEM excels in its first year

In November 2017, a Titan Krios cryo-electron microscope (cryo-EM) was inaugurated at the ESRF, the European Synchrotron, France. Data collected on this cryo-EM features in a Nature publication describing the activation cycle of a serotonin receptor, which is targeted by medication against chemotherapy- and radiotherapy-induced nausea.

“This publication is a true reward for us: the first one in less than a year from inauguration and we hope this kind of rewards will grow in number”, explains Isai Kandiah, ESRF scientist who runs the facility. “It shows the revolution that cryo-EM is leading in structural biology”, she adds. Thanks to cryo-EM, researchers can now freeze biomolecules, including membrane proteins of high medical importance, in several different conformations in action and visualise each of these to atomic resolution. Cryo-EM thus allows researchers to produce snapshots revealing the dynamics of proteins when they interact with other molecules, information that is crucial both for a basic understanding of life’s chemistry and for the development of pharmaceuticals. The user programme of the cryo-electron microscope at the ESRF is run jointly with the European Molecular Biology Laboratory (EMBL), the Institut de Biologie Structurale (IBS) and the Institut Laue-Langevin (ILL).

The research in Nature is a result of an international collaboration of scientists from the Institute of Structural biology (IBS-mixed research unit CEA-CNRS-University Grenoble Alps), CEA, CNRS, the Institut Pasteur, the University of Lorraine (France), the University of Copenhagen (Denmark), the University of Illinois (US) and the biotech company Theranyx. The focus of the paper, featuring data from the ESRF cryo-EM, is the activation cycle of the 5-HT3 receptor, belonging to the family of serotonin receptors. These receptors are well-known because they influence various biological and neurological processes such as anxiety, appetite, mood, nausea, sleep and thermoregulation, among others. Unlike the other serotonin receptors, which are G protein-coupled receptors, 5-HT3 is a neurotransmitter-gated ion channel and changes its conformation during activation. It is present in the brain, as well as in the enteric nervous system, the peripheral nervous system that drives the digestive tract.

>Read more on the European Synchrotron website

Image: A close-up view of the Cryo-EM at the ESRF.
Credit: S. Candé.