Tag Archives: biology

Cool views of molecular machines

Carolyn Moores, Professor of Structural Biology, writes about how academics in the Institute of Structural and Molecular Biology (ISMB) are using their new cryo-electron microscope to address big questions about antibiotic resistance, disease and life itself.

The state-of-the-art cryo-electron microscope at Birkbeck

Tucked discreetly in a corner of the Malet Street Extension building, a new state-of-the-art microscope is generating Terabytes of high-resolution imaging data. This £4M cryo-electron microscope (cryo-EM) is using Nobel Prize-winning technology to address a wide range of biologically and biomedically important questions. Researchers in the Institute of Structural and Molecular Biology (ISMB) are using it to study the molecular machines that are found within all living cells and that are essential for life. The ISMB – a joint research institute between Birkbeck and UCL – is internationally recognised as a centre of excellence for cryo-EM and for our research focused on diverse molecular machines.

In 2016, we were thrilled to be awarded a grant by Wellcome which, together with contributions from Birkbeck and UCL, funded the purchase of our Titan Krios microscope. Delivery day, earlier this year, was a big moment for the whole team. Prior to its arrival, we had been working hard with colleagues from Estates and external contractors to prepare a high-spec room for its arrival. Our microscope travelled in its own juggernaut from the Thermo Fisher factory in The Netherlands, with its ~3,300 parts carefully catalogued and packed in 21 crates. With nerves jangling, we all held our breath as the largest and heaviest part of the microscope was squeezed into the lift, travelled one floor to the basement without mishap, and edged its way out again. Constructed by dedicated engineers over the course of 8 weeks like a very big ship in a bottle, this 4.5 tonne, 4m tall monolith is now steadily producing up to 4TB of data a day.

Christmas came early – delivery of the cyro-electron microscope

Since its invention in the 1930s, electron microscopy has steadily improved in power and sophistication, and recent advances have moved this method to the forefront of structural biology. In particular, rapid freezing of samples to liquid nitrogen temperatures (~-195°C) allows sample preservation to be maintained inside the microscope, and provides some cryo-protection from the otherwise damaging effects of the electrons that are used for sample imaging. The cryo-EM field has undergone a technological revolution in the last ~5 years such that the application of this methodology has expanded enormously. The 2017 Nobel Prize in Chemistry was awarded to three pioneers in the field.

Our new microscope brings many benefits. First, it is very stable: since the ultimate goal of our experiments is to visualize the position and organization of all the atoms from which molecular machinery is built, microscope stability is vital. Coupled to this, the Titan Krios is designed to allow automated data collection such that, after a few hours of set up, data collection experiments can be left to run for several days without user intervention. Previous generation equipment required trained microscopists to sit at the microscope for as many hours as they could manage. Since our structural experiments require many thousands of sample images to be computationally combined to reveal their 3D structure, this is gruelling. Implementation of sophisticated data collection software is much more efficient and strongly preferred by the microscopists! In addition, although still requiring expertise and training, the Titan Krios is user-friendly while not compromising on its high-end capabilities. Around a dozen ISMB researchers have already received training and are busy collecting data for studies on topics as diverse as cancer, dementia, antibiotic resistance and malaria. Understanding the structures of biological molecules and assemblies reveals how they work and makes it possible to design drugs or treatments for diseases.

Installation in Birkbeck’s Malet Street building

With new opportunities come fresh challenges. Because the new microscope produces high-quality data – and a lot of it – so efficiently, our focus has now shifted to the problem of producing high-quality samples that can be fed into the Titan Krios imaging pipeline. This now represents a major bottleneck for many projects, both because of the time intrinsic to sample optimisation and because researchers need to learn the skills in order to undertake this optimisation. We are now focusing on ways to allow more projects and more researchers at UCL and Birkbeck to benefit from the cryo-EM “resolution revolution”.


Fostering collaborations between the UK and India – the way ahead for TB drug discovery research

This post was contributed by Arundhati Maitra, Associate Research Fellow at Birkbeck

Tuberculosis (TB) has re-emerged as a serious public health threat worldwide because of an alarming increase in the mortality rates due to drug resistant Mycobacterium tuberculosis strains and a deadly liaison between HIV and M. tuberculosis infection. There is an urgent need for identifying and validating new therapeutic leads to facilitate the development of novel anti-TB drug treatment.

An important element in intracellular survival and consequent pathogenesis of M. tuberculosis is its distinctive cell wall, of which peptidoglycan is a major structural, functional and regulatory component. The cytoplasmic steps of the biosynthesis of peptidoglycan are catalysed by a series of ATP-dependent ligases and they play a pivotal role by utilising ATP while incorporating specific amino acids sequentially to the C-terminus of the stem peptide; steps critical for cell wall cross-linking. They share a similar reaction mechanism and are essential for the growth of M. tuberculosis. As the reactions catalysed by these enzymes provide key precursors for the cell wall biogenesis and recycling, they are therefore considered as excellent therapeutic targets at the different physiological stages of the TB pathogen.

On a recent trip to India, Dr Sanjib Bhakta, Director of Mycobacteria Research Laboratory (MRL) part of the Institute of Structural and Molecular Biology, Birkbeck, University of London and UCL, visited a number of universities, specialised research institutes, organisations and schools to shed light on the world-class research being carried out at MRL researchers in the field of tuberculosis  drug discovery and to discuss new research and educational initiatives with India.

Dr Bhakta speaks to the Department of Molecular Biology and Biotechnology, Tezpur University (India)

Dr Bhakta speaks to the Department of Molecular Biology and Biotechnology, Tezpur University (India)

Dr Bhakta was invited by the erstwhile Vice-Chancellor of Dibrugarh University, Assam, India Professor Alak K. Buragohain, to engage in an interactive session with the research students of the Department of Molecular Biology and Biotechnology (MBBT), Tezpur University as well as the senior professors of the Department of Pharmaceutical Sciences, Dibrugarh University. A number of intense interactive sessions, held between July 23 – 25 2013 at the university departments was attended by the department’s students and faculty members and also by those in the Department of Chemical Sciences and Food Engineering and Technology. Dr Bhakta spoke about ‘Tackling drug resistance and persistence in Mycobacterium tuberculosis: integrative inter-disciplinary approaches in novel therapeutic intervention’, in the course of which he discussed whole cell assay techniques developed exclusively in his laboratory to evaluate potential anti-infective molecules. Highlighting some of the current work being carried out in his laboratory Dr Bhakta spoke about understanding ligand-protein interaction to unveil the mechanism of action of anti-mycobacterial compounds. He laid special emphasis on the need for an inter-disciplinary approach to fight TB and leprosy. A meeting with Professor Mihir Kanti Chaudhuri, Vice Chancellor, Tezpur University and other senior professors culminated in a proposal to forge collaborations between Birkbeck and Tezpur University, Assam, India. Any collective work between these two universities will benefit from the availability of potential anti-tubercular agents of plant origin harboured in the highly bio-diverse forests of Assam and the technical expertise of Dr Bhakta in testing the effectiveness of these agents in a wet lab setting.

Another invited seminar and one-to-one research meeting at which Dr Bhakta spoke was at the National Institute for Research in Tuberculosis, Chennai (India). Collaboration between NIRT and MRL is not new, as a materials transfer agreement already exists between the two world leading institutions. A memorandum of understanding currently being drafted will expectedly consolidate the existing relations.

Guest of Honour at the IDF Grants and Awards Function, Chennai (India)

Guest of Honour at the IDF Grants and Awards Function, Chennai (India)

A combination of social responsibilities, university outreach activities and common academic interests led Dr Bhakta to engage with the Indian Development Foundation (IDF). Established in 2005, the IDF is a non-government organisation with a three-fold goal to cater to education, development and health in the rural parts of India. Having been monumental in the elimination of leprosy in India, the organisation has moved its focus to tuberculosis. IDF hosted its Annual Grants Release Function on August 7, 2013 at Bharatiya Vidya Bhavan, Mylapore, Chennai at which Dr Bhakta was invited as a Guest of Honour to recognise the efforts of IDF and emphasise the need for continued support to such organisations. The annual feature is conducted to share resources with leprosy/TB projects and recognise schools in the region for their participation in social work and raising awareness of the cause. It was well attended this year with five-hundred audience members and renowned speakers on the panel including Mr. B.S. Raghavan (former Policy Advisor to the UN) and Dr A.R.K. Pillai (Founder President of IDF) and Mr. J. Ravichandran (CEO, German Leprosy and TB Relief Association) to name a few. The event was covered by Indian national television and print media units.

In an effort to stir the minds of tomorrow’s researchers, Dr Bhakta also accepted the opportunity to deliver a motivational talk at Maharishi School to a group of 50 A-level equivalent students, addressing their queries and encouraging them to embark on the life of a research scientist.


Alpha-1 Antitrypsin Deficiency – research update

Now almost a year into a two-year project, Dr Bibek Gooptu, from the Institute for Structural and Molecular Biology (ISMB) at Birkbeck and UCL, and his team are researching new treatments for the condition Alpha-1 Antitrypsin Deficiency and helping to advance our understanding of the mechanisms behind the condition.

The protein Alpha-1 Antitrypsin is created in the liver and then carried in the blood to the lungs, where it protects lung tissue. In people with Alpha-1 Antitrypsin Deficiency, a mutant gene within the liver cells causes the Antitrypsin protein to adopt a different structure, and individual protein molecules link together in chains, known as polymers. The liver is not able to secrete these polymers and so they remain stuck inside liver cells, damaging them. It also means that not enough Antitrypsin reaches the lungs from the bloodstream. People with Antitrypsin Deficiency are likely to suffer both from liver disease (e.g. cirrhosis or cancer of the liver) and, if they are smokers, they are likely to develop emphysema at a much younger age than someone without Antitrypsin Deficiency – probably in their 30s or 40s.

Promising compounds

Dr Gooptu and the project team (Gooptu group and collaborators at Birkbeck, UCL and Cambridge) have been studying compounds which will bind to the Antitrypsin protein in such a way that it is prevented from forming polymers. Because they have previously solved the molecular structure of the Antitrypsin in atomic detail they have identified three possible drug binding sites to target with new treatments. They have confirmed that small molecules (drug building blocks) predicted to target these sites in computer simulations bind the protein when tested experimentally in solution. The team are currently finalising computer simulations identifying a set of 750 compounds with similar binding characteristics to these ‘hits’. They will use these to test run the technology they are developing to assess the effects of many molecules at a time, not just on the protein in a test tube (which they do using a technique called mass spectrometry) but also at the site where the disease starts: in cells.

Example of a compound (cyan) successfully binding one of the target sites (surface features shown in grey) in Alpha-1 Antitrypsin (molecular structure shown as coloured ribbons) in computer simulation.

Example of a compound (cyan) successfully binding one of the target sites (surface features shown in grey) in Alpha-1 Antitrypsin (molecular structure shown as coloured ribbons) in computer simulation.

Testing in mammalian cells

Although a compound may bind to a protein in a solution, to be a successful drug the compound has to be able to enter the cell effectively. Once inside it must bind effectively in conditions that may be quite different. To see whether the compounds which bound successfully in solution can bind in the same way in cells like liver cells the team are testing them in cells known as CHO cells.  These are simple mammalian cells that grow well in a nutrient broth (cell culture) and behave like liver cells in terms of how they deal with normal Antitrypsin (secreting it efficiently) and disease mutant forms (accumulating polymers within the cell). It is already possible to check these cells for beneficial effects of drug-like compounds by checking whether the amount of secreted Antitrypsin goes up and/or the polymer levels go down. However at early stages of drug development the effects of the compounds may be more subtle, so Dr Gooptu and his post-doctoral researcher, Dr Nyon, are working with the Thalassinos group at UCL to identify more sensitive markers. For this they are working out the molecular signatures of health and disease in the part of the cell where proteins are prepared for secretion and where Antitrypsin polymers form and get stuck. These signatures are the levels of different proteins found within this area, known as the endoplasmic reticulum or ER for short. Not only is this useful for the current project, the signatures may themselves provide clues for other treatment options in the future.

Other proteins can aggravate or lessen disease

As well as the Antitrypsin protein there are hundreds of other proteins present in the ER. The amounts in which these proteins are present change depending on whether the cell is healthy or unhealthy. Some drop to undetectable levels while some new proteins are only found in one or other situation. Dr Gooptu compares this to looking at a street from an aerial view. In one view there are people in the street, with music and bunting. This is a street party and the equivalent to the ER of a healthy cell. In another view of the same street there might again be people in the street, but this time you also observe flames, fire engines and hoses. This is an emergency situation and the equivalent of the ER in an unhealthy cell. However, it is necessary to establish whether the people (proteins) in the second scene are causing the disease (arsonists) or fighting it (fire fighters). This will allow comparison of desirable with harmful responses to promising compounds and identify other proteins that might themselves be useful targets for future drug treatments.

Understanding how compounds are binding

Once they have identified the best compounds from the original set of 750 that bind Antitrypsin both in free solution and within cells, a technique known as Nuclear Magnetic Resonance (NMR) spectroscopy will be used to look very closely at the binding process and discover exactly how it takes place.

Binding of the Antitrypsin protein (grey ribbon representation) to a prototype drug molecule can be followed by NMR looking at changes seen when the interaction occurs around the structure at many individual points (spheres). In this case the colour coding shows many areas that change a lot (blue), whilst a few areas are stable (white)


Binding of the Antitrypsin protein (grey ribbon representation) to a prototype drug molecule can be followed by NMR looking at changes seen when the interaction occurs around the structure at many individual points (spheres). In this case the colour coding shows many areas that change a lot (blue), whilst a few areas are stable (white)



Using this technique the team can identify the characteristics of the protein:compound interaction. Armed with this information they can return to a library of thousands of compounds and identify further potential binders for testing and develop compounds which will bind in the most effective way.

Improving understanding of Alpha-1 Antitrypsin deficiency and the disease mechanisms

The approach taken by Dr Gooptu has developed existing methods and combined them in a new way. Usually drug companies will blind-test thousands of compounds in solution. Using the computational modelling before the in-solution testing meant that the team could identify a smaller number of compounds which were more likely to give positive results. The more detailed NMR spectroscopy studies are then targeted on a small number of compounds that show the most promise both in solution and in cells. This approach also has the additional advantage that while identifying compounds that bind Antitrypsin it also reveals more about how the genetic mutation causes disease in terms of harmful changes in proteins and cell responses.

Next steps

Having carefully developed the individual computational, mass spectrometry, cell biology and NMR techniques over the last year, in the next 12 months the project team will put them together and see how well they work in a pipeline for assessing the 750 test compounds. If this works well the pipeline could then be boosted to screen far greater numbers of compounds in future. However above the molecular scale, the project has already paved the way for bigger drug discovery projects in Alpha-1 Antitrypsin deficiency. Through his work, and the forum of research meetings convened by the US patient charity that funds the project (the Alpha-1 Foundation), Dr Gooptu has established contact with other groups working on a range of other approaches to identify new treatments. He is now collaborating with these groups and the Foundation to develop larger screens in which hit molecules can be rapidly identified from cell screening. The hits will then be studied in parallel by his group in Birkbeck and US research groups with complementary expertise to establish how they work and so how they can be further improved as efficiently as possible.

Dr Bibek Gooptu has been working on Antitrypsin Deficiency at Birkbeck since 2006. He is also a practising Consultant in Respiratory Medicine.  His research is supported by the Alpha-1 Foundation, the Medical Research Council and the Wellcome Trust.