Disentangling the neuromolecular networks involved in speech and language

As a biochemist/biophysicist working primarily with proteins, I am naturally drawn to the mechanisms of interactions of biological macromolecules. My name is Sylvia Fanucchi, and I am a senior Lecturer in the Protein Structure Function Research Unit (PSFRU) at the University of the Witwatersrand (Wits) in South Africa. I am interested in how things work at the molecular and atomic level, and how the structure of macromolecules leads to their function. This has inspired my research for the past six years which involves disentangling the neuromolecular networks involved in speech and language. With the GCRF START grant, the doors to collecting the detailed structural information we need through studying and obtaining crystal structures, have been opened for groups in Africa like ours. I have had multiple opportunities, thanks to the grant, to send crystals to the UK’s world class national synchrotron, Diamond Light Source (Diamond).

Senior Lecturer, Dr Sylvia Fanucchi, in the Protein Structure Function Research Unit at the University of the Witwatersrand in South Africa. Photo credit: Sylvia Fanucchi. ©Diamond Light Source

My research appeals so much to me because our ability to speak, to think, to read is fundamental to humankind. Indeed, my research question started with “what defines speech and language?” but has since expanded to include questions about cognition, reading, and a number of disorders associated with these such as Autism, Dyslexia, Epilepsy and Schizophrenia. Dyslexia, for example, occurs in at least one in 10 people world-wide, putting more than 700 million children and adults worldwide at risk of life-long illiteracy and social exclusion.[1].

Globally, it is estimated that one in 160 children has an Autism Spectrum Disorder (ASD), although estimates vary significantly across studies, and between developed[2] and developing countries[3]. In Southern Africa, very little is known about the prevalence ASD and it is understood that many cases go undiagnosed. Because these disorders can be debilitating and impact on the quality of life of those living with these disorders, and because there is currently no known cure, it is of utmost importance that the complex neuromolecular mechanisms that define these disorders be explored and far better understood. It is hoped that through our research more robust forms of therapeutics could be developed.

Piecing together neuromolecular complexes and networks

In my work, I investigate protein-protein and protein-nucleic acid interactions and try to piece together the neuromolecular complexes and networks that form in both space and time to better understand the mechanism of their interaction and how this is associated with language and cognition, as well as how changes in these may lead to certain disorders. I use an array of biochemical and biophysical techniques to achieve this study and the most prominent of these include, fluorescence anisotropy, isothermal titration calorimetry, hydrogen exchange mass spectrometry and single molecule kinetics. These techniques are used to study binding kinetics and thermodynamics as well as the dynamics and motions of molecules and their interactions with each other, such as the interaction between a protein and DNA, or the interaction between two proteins. The work we have done has mostly been conducted at the PSFRU but I also have collaborations with Dr Previn Naicker and Dr Stoyan Stoychev at the CSIR in Pretoria who assist with mass spectroscopy, and Dr Carlos Penedo[4] from the University of St Andrews in Scotland, UK, who assists with single molecule studies.

The promising biophysical studies conducted in this work will benefit greatly from detailed structural information, provided currently through access to Diamond with the GCRF START grant. Knowledge of the structures of macromolecular complexes allows us to fully understand our system at a level of detail that is otherwise unattainable. And this information, in addition to the dynamics/thermodynamics/kinetics data, will enable us to understand the complexity of these networks at atomic resolution. This will foster a deeper understanding of these mechanisms and enable detailed therapeutics to be designed that would help regulate these disorders, particularly if single strong contributing factors could be identified. Crystallography and solving the structures of the individual interacting partners, as well as of the complexes is therefore of fundamental importance in this project.

FOXP2 protein – the “the language gene”

The protein that sparked this investigation is a transcription factor (a protein that regulates the expression of genes) called FOXP2. FOXP2 was dubbed “the language gene” in the early 2000s when a mutation in this gene that severely impeded DNA binding was found to result in a form of verbal dyspraxia in a family of individuals in the UK. Because FOXP2 is a transcription factor, it is located in the nucleus of cells and its role is to bind to the promotor region of certain genes and facilitate/regulate their transcription to mRNA which ultimately results in the translation and thus expression of that particular protein. Therefore, the regulation of transcription (and hence translation) of any protein will have a direct effect on the functioning of that protein. Our focus is thus on transcriptional regulation because it is the process that initiates downstream effects and predicts which genes will be turned on or off and hence, crudely put, controls the way we function.

Over the past three years, this project therefore focused on the mechanism of DNA binding of FOXP2. Through this work we were able to meticulously describe what drives the interaction of the DNA-binding domain with cognate DNA. We identified electrostatic interactions that played a critical role, we studied binding sequences to gain insight into binding specificity and affinity, we outlined how a domain-swapped dimerisation event that is unique to this subfamily of FOX proteins was able to assist in the dynamics of the DNA binding event, and we described the thermodynamic and kinetic events that occur during binding. In essence, by describing how the transcription factor interacted with DNA, we were able to tell which sequences it preferred, which conditions were most favourable for transcription, and how the structure and fold of the protein was necessary for transcription to occur. Knowing this helps us understand what is necessary for certain genes to be turned on or turned off and how we could interfere with this process.

The focus of our work then moved from DNA binding to the complex network of neuromolecular protein-protein interactions and we began to piece together other interacting partners of FOXP2 and how these interactions affected transcriptional regulation. One specific interaction has yielded a very interesting link to Autism that we are currently exploring further. I have established a very fruitful collaboration with Dr Carlos Penedo from the University of St Andrews primarily through a Newton Fellowship from the Royal Society and the single molecule work done through this collaboration has helped to resolve intricate details about these interactions that I am very excited about[5].

Fast and remote access to diffraction and data collection with Diamond and the GCRF START grant

The opportunities to send crystals to Diamond with the assistance of the GCRF START grant have been revolutionary in enabling us to have fast and remote access to diffraction and data collection that would otherwise have been logistically far more difficult to achieve, and therefore far more sparsely accomplished. Unfortunately, up to now, crystallisation in this project has been a challenge and so far, we are yet to achieve the crystals we require. The fact that the proteins we are attempting to crystallise have not yet had their structures solved and published in the Global Protein Data Bank (PDB) attests to the challenge we knew we would face in obtaining good diffraction data. And while we have obtained some crystals successfully, none of the data we have obtained has been worthy of solving the structure. Nevertheless, the fact that we can obtain crystals is promising and I am determined to persevere with this work until we are successful.

Aerial view of the UK’s national synchrotron, Diamond Light Source at the Harwell Campus in Oxfordshire
©Diamond Light Source

My students are all working on aspects of this project. They work on protein-protein interactions, crystallisation and structural biology, biophysics, and biochemistry. I am currently supervising 5 PhD students and 4 MSc students from diverse backgrounds, 8 of whom are females. I also have 2 postdoctoral Fellows – one from Lesotho and one from Kenya – that have worked under me for the past two years.  In 2020, I graduated 2 PhD students and 2 MSc students. The students are given the autonomy to operate equipment, design experiments and analyse their data. Where possible, and covid-19 pandemic permitting, I encourage them to participate in international workshops and to visit other labs to gain experience and exposure.

Riyaadh Mayet is one of the early career MSc Students in my team.

“The GCRF START grant has enabled the African continent to foster development in synchrotron techniques through collaboration with the UK.” she says. “My MSc project deals with the structural biology of DNA-binding by the TBR1 T-box transcription factor implicated in Autism. The grant has enabled me to send my samples to the Diamond Light Source for diffraction, and without it, it would be very difficult if not impossible to obtain such data. It has also taught me how to better collaborate with fellow researchers, as well as given me the opportunity to learn how to diffract crystals to obtain atomic resolution data. Lastly, I have indirectly benefitted through learning about protein crystallography from fellow researchers who have used the START grant.”

Dr Sylvia Fanucchi with some members of her team of researchers at the Protein Structure Function Research Unit, at the University of the Witwatersrand in South Africa. From left: Ilan Kirkel, Joni Symon, Dr Ashleigh Blane, Heather Donald, Dr Sylvia Fanucchi, Dr Monare Thulo, Riyaadh Mayet and Aasiya Lakhi.
Photo credit: Sylvia Fanucchi. ©Diamond Light Source

Despite the challenges we have encountered with solving structures in this project, I am very grateful for the support received through the GCRF START grant and the support structures put in place – in particular, in bringing together a strong network of South African crystallographers. Knowing that I have this group of colleagues available across the country that forms a support structure is very reassuring. I am confident that the assistance and opportunities offered to me by this grant over the years is going to result in the ultimate success of my crystal structure dreams for this project and I am both grateful and excited for what the future holds.

Commenting on Sylvia’s research project and the access to infrastructure such as the Diamond synchrotron, Head of School of Molecular and Cell Biology (MCB), Prof Marianne J. Cronje, says,

“I am wholly in support of these research endeavours. Dr Sylvia Fanucchi is an incredibly talented researcher and her position within the school’s Protein Structure Function Research Unit strengthens her efforts by providing access to high-end research infrastructure to support her research in the school.”

Senior Lecturer, Dr Sylvia Fanucchi, in the laboratory at the Protein Structure Function Research Unit, University of the Witwatersrand in South Africa.
Photo credit: Sylvia Fanucchi. Diamond Light Source

Addressing disability is referenced in many of the UN’s Sustainable Development Goals (SDGs), namely health, education, economic growth and employment, inequality, accessibility of human settlements, and others.  Read more about the SDGs here.

[1] https://www.dyslexia-and-literacy.international/wp-content/uploads/2016/04/DI-Duke-Report-final-4-29-14.pdf accessed 20.02.2021

[2] In the USA~10% of school children struggle with dyslexia and ~1 of every 59 school children is diagnosed with ASD (Yuhang Lin et al Int. J. Environ. Res. Public Health 2020, 17, 7140). The number with ASD has increased over the years reflecting both an increase in awareness as well as a potential increase in environmental triggers although the disorder is currently believed to be predominantly genetically determined.

[3] https://www.who.int/news-room/fact-sheets/detail/Autism-spectrum-disorders (accessed 20.02.2021)

[4] https://risweb.st-andrews.ac.uk/portal/en/persons/carlos-penedo(e1903c4a-e36d-4555-9c7e-e604c9873ba0).html

[5] Thulo M, Rabie MA, Pahad N, Donald HL, Blane AA, Perumal CM, Penedo JC, Fanucchi S. Biosci Rep. 2021 Jan 29;41(1):BSR20202128. doi: 10.1042/BSR20202128

Promising first steps towards an inhibitor targeting the South African HIV-1 subtype C protease (C-SA PR)

In a bid to design clinical drugs to improve health outcomes for people living with a particular strain of HIV and its mutants, scientists at the University of the Witwatersrand’s Protein Structure-Function Research Unit (PSFRU) in South Africa have embarked on a clinical drug discovery journey with promising results. The aim of the research, which receives funding from the GCRF START grant, is to develop a novel inhibitor specifically designed to target the South African HIV-1 subtype C protease (C-SA PR) and its mutants.

The purpose of developing an inhibitor is to stop C-SA PR’s activity by preventing the formation of mature copies of the human immunodeficiency virus (HIV). This would improve health outcomes in line with Sustainable Development goals across South Africa by increasing drug efficacy, reducing adverse side effects and drug resistance, as well as benefitting populations infected with the HIV subtype C in other sub-Saharan countries, India, China and Brazil[1].

GCRF START Post-Doctoral Research Fellow, Dr Ramesh Pandian (L), discussing crystallisation set up with GCRF START Co-I, Prof. Yasien Sayed (R), for the screening of crystals at the Protein Structure-Function Research Unit, University of the Witwatersrand, South Africa. Photo credit: Ramesh Pandian. ©Diamond Light Source

There is no permanent cure for HIV/AIDS which has claimed the lives of an estimated 32.7 million people globally since the beginning of the HIV pandemic (UNAIDS, 2020[2]). A number of drugs have been developed and approved by the USA’s Food and Drug Association (FDA) to increase the quality and duration of life in HIV infected individuals in some parts of the world. However, these are not specific to the HIV-1 protease subtype C which is dominant in South Africa – a country with approximately 20% of the global HIV infection rate and 10.44% of the global AIDS-related deaths (UNAIDS, 2020)[3].

To date, the scientists at the PSFRU have screened ten drugs in the FDA approved and Zinc drug databases, with seven hits that show promise for optimisation as inhibitors. They have solved one protein structure through sophisticated computational modelling, and with high-resolution X-ray crystallography data collected on beamline i03 at the UK’s national synchrotron – Diamond Light Source (Diamond) – have solved the structure of the South African HIV-1 subtype C protease (C-SA PR), the results of which were deposited in the global Protein Data Bank (6I45.pdb) in 2018 (Fig.1).

Fig.1 Ribbon structural representations of the HIV-1 subtype C-SA PR (cyan) and a mutant protease (purple). The structure of the mutant protease was determined using diffraction data obtained at the UK’s national synchrotron, Diamond Light Source (Diamond). PDB ID: 6I45. Sherry, D., Pandian, R., Achilonu, I.A., Dirr, H.W., Sayed, Y.  (2018), Crystal structure of I13V/I62V/V77I South African HIV-1 subtype C protease containing a D25A mutation. DOI: 10.2210/pdb6I45/pdb[4]; PyMOL Molecular Graphics System (Schrödinger LLC., Portland, USA[5]).

The research involves characterising the structure and function of the South African HIV-1 subtype C protease (C-SA PR) and its mutants using state-of-the-art computational and experimental methods made possible by the GCRF START grant. The scientists want to understand what role amino acid insertions and mutations the HIV-1 protease may have on clinical drug binding so they can design an effective inhibitor.

This research builds on a previous study[6], in which a blood sample was taken from a drug naive infant born to an HIV positive mother. To prevent the transmission of the virus to her baby, the mother had received reverse transcript inhibitor treatment (but not protease inhibitors) prior to the birth. However, when the baby was born it was found to be HIV positive and a mutation was present rendering current drug therapy ineffective.

“It is the results like this of other research on the South African HIV-1 C-SA PR, and the impact of the disease on individual lives and livelihoods, which drives our motivation. The fact that the baby had developed drug resistance mutations is very rare in mother-to-child transmission but no less concerning,” says Professor Yasien Sayed, who heads up the PSFRU and leads the research on the HIV-1 C-SA PR, “and there is evidence that some adults are also failing drug therapies. We therefore need to develop treatments which work more effectively against the HIV-1 C-SA PR and its mutants if we are going to improve clinical outcomes for the large population of HIV positive adults and children in South Africa, and further afield.”

Some of the first steps in the team’s drug discovery journey have been computational involving Molecular Dynamics Simulation[7] of the HIV wild type C-SA PR and its mutants. After this, drugs in various databases were screened[8].  As a result, the scientists found a promising drug from the FDA database that binds with the HIV wild type C-SA PR and its mutant with best docking scores and energies.

“We modelled the homology structures of the HIV wild type protease and its mutants using the template South African wild type HIV-1 Subtype C Protease (PDB ID: 3U71),” explains Dr Pandian who is a Post-Doctoral Research Fellow funded by the GCRF START grant specialising in the computational aspects of the research. “The structure was solved at 2.72Å using the software packages: Swiss model/modeler/I-Tasser, followed by experimental validation of the modelled structures with in-house computer software. We are excited by the preliminary results, which are better than the current FDA approved drugs, although the computational results now have to be proved through wet lab experiments, along with the best results from the screened from the Zinc database.”

Dr Ramesh Pandian, GCRF START Post-Doctoral Research Fellow at the University of the Witwatersrand, South Africa, in the process of solving of a crystal protein structure. Photo credit: Ramesh Pandian. ©Diamond Light Source

To conduct these studies, the PSFRU has its own computational and wet lab facilities for Molecular Dynamics simulation, docking studies, protein expression and purification. Screening of crystals is carried out using an Oryx8Protein Crystallization Robot (Douglas Instruments, UK) and testing of crystals using a home-source X-ray diffractometer. However, synchrotron facilities are not available on the African continent, so access to the Diamond Light Source synchrotron (Beamlines i03, i04, and also i04-1 and i24) is achieved remotely from the PSFRU lab in South Africa in order to characterise the structure and function of proteins at high resolution.

“Having access through the GCRF START grant to experimental synchrotron techniques like X-Ray crystallography at Diamond to obtain crystals and to solve the structures at high resolutions has been revolutionary for us,” reports Dr Pandian. “It is ultimately the combination of computational and experimental techniques that makes it possible to see how well the drugs are binding to optimise them for the South African HIV-1 subtype C protease (C-SA PR)[9].

“During the wet lab experiments,” Dr Pandian continues, “we can’t screen the whole drug library for the target protein and it’s very costly to purchase the drugs for screening. The theoretical part of the drug discovery method is therefore useful for generating three dimensional structures for any proteins when the crystal structures are not available in the PDB databases, and for sorting out the best ligand / inhibitors for the protein target before starting protein characterisation wet lab experiments.”

Aerial view of the UK’s national synchrotron, Diamond Light Source (Diamond) on the Harwell Campus, in Oxfordshire, UK. ©Diamond Light Source

Scientific results are not the only progress being made by the PSFRU team in their research which is meeting the UN’s Sustainable Development Goals for health (SDG 3); great strides have also been made in the PSFRU in terms of education and capacity building in structural biology (SDG 4) with more than 30 postgraduate students involved in the collaboration with the GCRF START grant since 2019. This includes Dr Ramesh Pandian, and Ms Mpho Setshedi who is a MSc. candidate working on the wet lab studies of the HIV-1 C-SA PR and its mutants.

“The research is meaningful,” says Ms Setshedi, “I feel like we are doing a good job and doing something to solve a challenge that impacts South Africa. I hope it contributes something big – an effective HIV inhibitor. In terms of what I am learning, there are challenges in this field but once you get the hang of the techniques you just need to persevere. Getting funding is a struggle in my field generally and there aren’t a lot of women doing this work. There have also been challenges caused by the COVID-19 lockdown in 2020 – but I haven’t let anything discourage me.”

Ms Mpho Setshedi, MSc. student at the Protein Structure-Function Research Unit, University of the Witwatersrand, South Africa. Photo credit: Mpho Setshedi. ©Diamond Light Source

Read more about the UN’s Sustainable Development Goals here

[1]: https://www.unaids.org/en/AIDS_SDGs accessed 3.3.2021

[2]https://www.unaids.org/en/resources/documents/2020/UNAIDS_FactSheet accessed 3.3.2021

[3] https://www.avert.org/professionals/hiv-around-world/sub-saharan-africa/south-africa accessed on 4 March 2021; For latest (2019) South African country statistics see: https://www.unaids.org/en/regionscountries/countries/southafrica

[4] https://www.rcsb.org/

[5] http://www.schroding-er.com ; Schrödinger, L. & DeLano, W., 2020. PyMOL, Available at: http://www.pymol.org/pymol

[6] https://pubmed.ncbi.nlm.nih.gov/30793914/ accessed 9.3.2021

[7]The Molecular Dynamics (MD) simulations of validated structures were performed under physiological conditions for 100ns using GROMACS software packages.  The MD simulated structures were analysed thoroughly and extracted the energy minimised structure for further analysis. Important parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radiation of gyration (Rg) and hydrogen bonding analysis were carried out.

[8]The energy minimised structures of HIV wild type and its mutants were used for the screening of drugs from different data bases such as the Zinc database and FDA approved drugs database.

[9]The results of the binding pocket analysis of the protease complex form obtained by the docking studies with best ligand directed the scientists to modify the side chain with the combination of different R group of the drug to improve the binding affinity.

Engineering sustainable solutions – Enzymes to tackle toxic waste

The effects of human activity on climate change are evident in accelerated changes to global climatic conditions. As a result, there have been efforts to decrease human impact on the environment. This includes sustainable and environmentally friendly waste management systems, bioremediation of damaged ecosystems and biocatalysis as a replacement for conventional chemical synthesis in industries in line with the UN’s Sustainable Development Goals.

One way to address these challenges is through the use of enzyme biotechnology in which proteins (enzymes) are used as industrial catalysts. Nitrilases are widely used enzymes with potential in the production of high-value fine chemicals including medicines, bioremediation and waste management. A problem with using enzymes in environmental remediation is that naturally occurring enzymes are susceptible to degradation and inactivation under harsh conditions. They can, however, be engineered to make them more tolerant of these conditions. Nitrilases have intrinsically robust, spiral structures that suggest that substantial improvements in stability are possible.

My name is Lenye Dlamini and my PhD study in the Structural Biology Research group at the University of Cape Town (UCT) concerns the engineering of a cyanide-degrading nitrilase enzyme that can be used to remediate cyanide waste in the textile, electroplating and gold-mining industries. In particular, cyanide is used in huge quantities in these industries and spills or unsafe disposal results in environmental degradation and causes harm (and occasionally death) to humans and livestock.

Lenye Dlamini, PhD student at the University of Cape Town, South Africa. Photo credit: Lenye Dlamini. ©Diamond Light Source

My study will use predictive biophysical methods based on structural knowledge of cyanide degrading nitrilases to measurably improve their tolerance of non-optimal temperature ranges and enhance the operational stability of the enzyme so that they can be routinely used for safe and cheap disposal of cyanide waste.  The project builds on the work of Dr Andani Mulelu[1],2 and several of Prof Michael Benedik’s students at Texas A&M University who used directed evolution, a technique in which random amino acid mutations are introduced throughout the protein, to increase protein stability and ultimately led to the first structure of an enzyme of this type. My goal is to use this structure to identify specific amino acid changes that will lead to increased stability.

I am working from Prof Trevor Sewell’s (GCRF START Co-I) laboratory at UCT and the Electron Microscope Unit at the Aaron Klug Centre for Imaging and Analysis. My work is a component of the work on nitrilases being done by a large team of local and international collaborators (my first experience of collaborating with scientists outside of South Africa) that includes in South Africa: Dr Jeremy Woodward (GCRF START Co-I), Dr Gerhard Venter and Prof Roger Hunter at UCT, Prof Dean Brady at the University of the Witwatersrand, Dr Nishal Pharbhoo at UNISA; in Germany: Prof Andreas Stolz and Mr Erik Eppinger at the University of Stuttgart, Prof Markus Piotrowski of the Ruhr University at Bochum, and Prof Achilleas Frangakis at Goethe-University, Frankfurt.

While the direct impact of my research project is environmental, it has broader implications in a variety of industries. There is growing demand for biological agents and processes that will replace conventional processes of managing waste and chemical synthesis. Enzymes, including the nitrilases, have taken centre-stage in this regard and present not just an environmentally benign alternative but one that produces better reaction products (they are highly specific in the enantiomers and regions on compounds that they bind to, leading to more specific reaction products).

It is critical that we understand and can adapt enzymes for these new uses.  Nitrile-containing compounds are widespread in nature and are also utilised in industries including agriculture, mining, pharmaceuticals and the plastics and paper industry. Most nitrile-containing compounds are toxic, mutagenic, and carcinogenic. We are trying to design a way that will result in the use of harmful compounds as substrates for the catalysis of useful compounds in a sustainable and environmentally benign manner.

In my research on nitrilases, I am building on previous research skills I gained through my MSc. project which also benefitted from access to the UK’s national synchrotron, Diamond Light Source (Diamond). Through supporting research, workshops, mentoring and supervision, and other aspects of these labs, the GCRF START grant has also, by extension, supported the development of my research career. In addition, workshops presented by structural biologists from Diamond in South Africa, have enabled me to acquire skills that include molecular biology techniques, protein crystallography, electron microscopy and data collection at synchrotrons.

My Master’s research was in rational drug design against Mycobacterium tuberculosis using molecular and structural biology techniques. One of the main outcomes of my study was the crystal structure of thiamine monophosphate kinase from Mycobacterium tuberculosis, solved at a resolution of 2.19 Å, with data collected using the i04 beamline at Diamond with access through the GCRF START grant.

Aerial view of the UK’s national synchrotron Diamond Light Source on the Harwell Campus in Oxfordshire. ©Diamond Light Source

Commenting on the research outlined above, Prof Trevor Sewell, Lenye’s supervisor, said,

“The work of the last 30 years has provided a wealth of knowledge about the structures, occurrence and chemistry of members of the ubiquitous nitrilase superfamily of enzymes. This has led to their widespread use as industrial enzymes and a recognition that some superfamily members are potential drug targets. Even so, our understanding of their mechanism and our ability to introduce desirable properties through design is very limited. Lenye’s work seeks to surmount the barriers, leading to the ability to enhance at least one property of the cyanide degrading nitrilases, the thermostability, by design and then verify that the desired goal has been achieved experimentally using CryoEM and differential scanning calorimetry.”

Find out more about the UN’s Sustainable Development Goals here

About Lenye Dlamini

Lenye Dlamini was born and raised in Mbabane, the capital city of Swaziland, which is also where she received her primary and high school education. Lenye was inspired into science at high school through her biology and chemistry teacher who, Lenye says, saw the potential in her and motivated her to work extra hard. Prior to embarking on her PhD studies, Lenye’s tertiary education was at the University of Pretoria in the laboratory of GCRF START Co-I, Prof Wolf-Dieter Schubert. Lenye is currently a PhD candidate in Medical Biochemistry in the Structural Biology Research group, Department of Integrative Biomedical Sciences, Faculty of Health Sciences at the University of Cape Town, South Africa.

[1] Mulelu, A. 2017. Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1. University of Cape Town. http://hdl.handle.net/11427/24446

2 Sewell, BT, Frangakis, A, Mulelu, A and Reitz, J. (2017). The structure of the cyanide dihydratase (CynD) from Bacillus pumilus. Acta Cryst.  A73, C1296. DOI: 10.1107/S2053273317082791

Focus on fungal oxidoreductases for infectious disease drug targets

“The atomic structure of proteins provides an intimate insight into these magnificent macromolecules. This knowledge is crucial to truly understand how they function; whether it is to answer a burning question or to manipulate them – either to enhance if their reactions are desirable, or to inhibit if they are harmful.”   

Prof. Dirk Opperman, University of the Free State, South Africa 

Our research studies the structures of bacterial and fungal oxidoreductases (enzymes) which are possible drug targets to combat infectious disease. The current focus is fungal drug targets for fungal infectious diseases which can be very serious, especially for immune-compromised patients, such as those who are HIV/AIDS positive, organ transplant receivers, patients undergoing chemotherapy, and many more. This research is performed at the University of the Free State’s (UFS’s) Department of Microbial, Biochemical and Food Biotechnology1 and is led by the two Principal Investigators (PIs) of the Biocatalysis group at UFS, Prof. Dirk Opperman, who is a GCRF START Co-Investigator (Co-I), and Prof. Martie Smit. In our laboratory, we have solved the structures of a number of bacterial and fungal enzymes by X-ray crystallography over the past few years, two of the most recent – solving the structures of fungal cytochrome P450 reductase (Dec 2019) and Baeyer-Villiger monooxygenase (Feb 2020) – were assisted by the GCRF START grant and published in the journals Scientific reports2 and Catalysts3 respectively, as described later in this article.   

Dr Carmien Tolmie conducting molecular biology experiments at the University of the Free State’s Department of Microbial,
Biochemical and Food Biotechnology. Photo credit: Rodolpho do Aido Machado. ©Diamond Light Source 

The scale of the Fungal infection and drug resistance challenge  

Currently, there are three classes of anti-fungal drugs that are used to combat infectious fungal disease, but there is an increasing number of drug resistant (and even multi-drug resistant) fungi against these drugs meaning that these pathogenic fungi have become or are becoming resistant to the current medication used to treat patients. If no drugs are effective against invasive opportunistic fungi, the prognosis for immune-compromised patients is very poor, and many people will die. 

“It is therefore imperative that we search for and develop new antifungal compounds to address the growing challenge [of drug-resistance to opportunistic fungi], which impacts countries across Africa, as well as globally. This is especially urgent if the world is to meet the UN’s Sustainable Development Goals of Health and Wellbeing and Food Security by 2030.” 

Dr Carmien Tolmie, University of the Free State, South Africa 

Fungal infections are often underreported and because of this the extent of the situation is not fully known.  In South Africa, this is of particular concern because of our high incidence of HIV/AIDS. For example, one group4 reported that 90 % of HIV/AIDS positive patients on prolonged treatment contract oropharyngeal candidiasis (also called Thrush), an infection caused by a yeast, which is a type of fungus called Candida  (Dos Santos Abrantes, McArthur and Africa 2014). However, this is only one statistic, and the problem is much wider. In another example, Cryptococcal meningitis is a deadly brain infection caused by the soil-dwelling fungus Cryptococcus. Worldwide, nearly 220,000 new cases of cryptococcal meningitis occur each year, resulting in 181,000 deaths, most of which occur in sub-Saharan Africa (CDC, 2020). 

Using the powerful beams of Diamond’s synchrotron light to determine protein structures 

Our quest to find new drug targets involves examining the chemical processes that happen in the fungal cell in order to keep the cell alive. We choose an enzyme – a special type of protein involved in these processes – which will be a good target for anti-fungal medication. The experiments we do to produce our protein crystals include molecular biology, protein expression, purification and crystallisation.  We clone the gene that encodes the enzyme and insert it into a suitable host to produce the protein, such as the bacterium Escherichia coli, by protein expression methods. Escherichia coli is easy to manipulate and inexpensive to culture in large volumes. We then isolate the enzyme by protein purification methods which exploit the physicochemical properties of the enzyme to separate the target from the host proteins, and crystallise it before we examine it by X-ray crystallography.  

Dr Carmien Tolmie purifying proteins using an AKTA chromatography system at the University of the Free State’s Department of Microbial,
Biochemical and Food Biotechnology, South Africa. Photo credit: Rodolpho do Aido Machado. ©Diamond Light Source 

We use an in-house crystallisation robot at UFS to prepare the crystallisation trials and we regularly collect crystal diffraction data via remote access at the macromolecular beamlines of the UK’s national synchrotron –  Diamond Light Source (Diamond). This is a great help as we can control the beamline equipment from our offices, so we don’t incur the expense of travelling to the UK to use synchrotron techniques essential for our research. While we have X-ray diffractometers at the University of the Free State, they are not nearly as powerful as the beamlines of the Diamond synchrotron. Diamond’s beamline hardware has been developed to such an advanced stage that data collection can proceed very rapidly, enabling us to collect data much faster (minutes) than at our home sources (days). This high throughput is essential when searching for and identify tiny molecules that might potentially bind to the protein and possibly act as inhibitors, as large libraries of fragments must be screened. 

“The brilliant light generated by Diamond (10 billion times brighter than the sun!)  enables us to determine the structure of the proteins to extremely high resolutions, as well as structures from small or weakly diffracting crystals that we cannot study with our own laboratory techniques.” 

Dr Carmien Tolmie, University of the Free State, South Africa 

 We use the protein structure to search for small molecules that will bind to the enzyme and possibly stop it from working (act as inhibitors). In Biocatalysis, knowledge of the protein structure can identify ways in which one can change, or mutate, the enzyme to perform the specific reactions desired.  If the protein is a drug target, the structure can be used in Structure-Based Drug Discovery to develop new medications. This process can also be used for other applications like developing new pesticides for agriculture. Therefore, the next step in the research process is to use fragment screening methods  to identify lead compounds that can be further developed into inhibitors, thus helping develop a next generation drug. The fragment screening is done in collaboration with Prof. Frank von Delft and the XChem group on the I04-1 beamline at Diamond through the GCRF START grant and will be undertaken once Covid19 travel restrictions are lifted.  

Beamline I04-1 Experimental Hutch – Sample changer and sample environment at the UK’s national synchrotron, Diamond Light Source. ©Diamond Light Source Ltd 

Solving the structures of fungal cytochrome P450 reductase and Baeyer-Villiger monooxygenase  

Recently, we were able to solve and gain new insights into the structures and mechanisms of the fungal cytochrome P450 reductase (CPR) from Candida tropicalis and the Baeyer-Villiger monooxygenase BVMOAFL210 from Aspergillus flavus, research that was made possible by the GCRF START grant. The results were published in the journals Scientific reports and Catalysts respectively, and the research on the CPR was done in collaboration with scientists from the University of Cape Town5, who are also part of the START project. The publications were co-authored by START PDRAs Ana Ebrecht (first author on CPR paper) and Rodolpho do Aido Machado (co-author on BVMOAFL210).  

The CPR plays a pivotal role in primary and secondary metabolism of different species, from bacteria to animals and plants. In fungi, it supplies electrons to enzymes that are vital for the survival of the organism. The CPR mechanism is complex and involves conformational changes that need to be finely tuned to optimise the process. The structural characterisation of the CPR helps to understand how this process occurs and what are the differences with the human homolog, opening the possibility to use it as a drug target. The structure and mutation data of BVMOAFL210 allowed us to better understand the role of the amino acid at a specific position in the enzyme, in terms of regioselectivity (the position in the substrate where the oxygen atom is inserted) as well as the sulfoxidation (the number of oxygen atoms inserted in a sulfur-containing compound). This residue may be used in future studies for directed evolution experiments to evolve the enzyme to catalyse a desired reaction.  

In order to achieve the results, we first needed to produce pure protein. The proteins were crystallised by the vapour-diffusion method with the Douglas Oryx Nano crystallization robot located in our crystallography lab in our department. In these experiments, a library of 384 crystallisation conditions were screened and a few conditions yielded crystals. These crystals were cryo-cooled and shipped at liquid nitrogen conditions in a specialised container to Diamond where we collected data on the macromolecular crystallography beamlines through remote access. We processed the data and solved the structure with programs from the CCP4 suite of macromolecular data processing software. The proteins were characterised further by investigating their kinetic properties with several spectrophotometric assays using a UV/Visible light spectrophotometer.  

Dr Carmien Tolmie using a Douglas Oryx Nano crystallisation robot to set up protein crystallisation trials at the University of the Free State’s Department of Microbial,
Biochemical and Food Biotechnology, South Africa. Credit Rodolpho do Aido Machado. ©Diamond Light Source 

For BVMOAFL210, we created mutations at a specific position and determined how these mutations alter the biocatalytic profile of the enzyme using whole-cell biotransformation experiments, followed by Gas-Chromatography Mass Spectrometry (GC-MS) analyses. In terms of the fungal cytochrome P450 reductase (CPR), the next steps will be to use the CPR for fragment screening to gain further, more detailed insights. This method uses protein crystals of the target enzyme to identify small molecule fragments that bind to the enzyme. These fragments are then elaborated into larger molecules with higher potency, which will hopefully not only inhibit the specific enzyme, but also the growth of pathogenic fungi.  

Benefitting from increased research capacity through the GCRF START grant 

The grant has contributed greatly to the research capacity of the Biocatalysis and Structural Biology Research group, making it possible to appoint a START Postdoctoral Research Assistant (PDRA) who focused on structural biology research, the research itself partially funded by START. START also helped to develop the skills of the researchers in the group by funding workshops, as well as workshop attendance, and a research exchange of the START PDRA to the UK in 2019. In addition, START has introduced us to world-class scientists at Diamond and other institutions who we can consult if we need advice on our experiments.  

“With this sharing of knowledge, capacity building and cutting-edge research enabled by the GCRF START grant, it is our fervent aim to make a lasting, positive impact in terms of sustainable health, well-being and food security solutions now, and well into the future.”

Dr Carmien Tolmie, University of the Free State, South Africa 

Click here to read more about the UN’s Sustainable Development Goals  


We would like to thank Prof. Trevor Sewell from the START Centre of Excellence at the University of Cape Town’s Aaron Klug Centre for Imaging and Analysis for the pivotal role he has played both in GCRF START and Structural Biology in South Africa. The START Centre of Excellence is a collaborative, shared resource where participants in the START programme can access everything they need to get started with their research, such as advice and the necessary equipment which may not be available in their own laboratories elsewhere. This also includes the technological support and expertise to access the UK’s national synchrotron – Diamond Light Source – through the GCRF START grant (such as support with sample preparation, shipping, and remote access experiments). 

Dr Carmien Tolmiehttps://orcid.org/0000-0001-9095-3048 

Dr Carmien Tolmie, researcher in the Department of Microbial, Biochemical and Food Biotechnology
at the University of the Free State, South Africa. ©Diamond Light Source 

Prof.Dirk Oppermanhttps://orcid.org/0000-0002-2737-8797 

Prof. Dirk Opperman, researcher in the Department of Microbial, Biochemical 
and Food Biotechnology at the University of the Free State, South Africa. ©Diamond Light Source 


[1] Research in the Department falls broadly into three main areas: (i) production of safe and novel food products, (ii) biocatalytic production of chemicals or bioremediation of chemical pollution, and (iii) improvement of human and animal health. Our Biocatalysis Group focuses heavily on biocatalysis which involves the use of one or more enzymes, either as cell-free enzymes or enzymes in whole cells, to convert a substrate into a value-added product. This includes converting alkanes, alcohols, fatty acids or monoterpenes into value added building blocks of pharmaceuticals, bio-plastics, cosmetics, flavours and/or fragrances.

[2] Ebrecht AC, Van der Bergh N, Harrison STL, Smit MS, Sewell BT and DJ Opperman (2019). Biochemical and structural insights into the cytochrome P450 reductase from Candida tropicalis. Scientific Reports 9:20088. doi: 10.1038/s41598-019-56516-6: https://www.nature.com/articles/s41598-019-56516-6

[3] Tolmie C,Do Aido Machado R, Ferroni FM, Smit MS and DJ Opperman (2020). Natural variation in the ‘control loop’ of BVMOAFL210 and its influence on regioselectivity and sulfoxidation. Catalysts 10(3): 339. doi: 10.3390/catal10030339: https://www.mdpi.com/2073-4344/10/3/339

[4] Dos Santos Abrantes PM, McArthur CP, Africa CWJ. Multi-drug resistant oral Candida species isolated from HIV-positive patients in South Africa and Cameroon. Diagn Microbiol Infect Dis 2014;79:222–7.

[5] https://www.news.uct.ac.za/article/-2020-03-12-sa-women-take-the-lead-in-structural-biology

New generation solutions to Neglected Tropical Diseases and Nosocomial ESKAPE Infections

“Africa is a bank when it comes to diseases. The beauty of the GCRF START grant is that it offers us the opportunity to deal with these challenges in Africa, while enabling international collaboration and access to amazing synchrotron facilities with world class results.”

Dr Ikechukwu Anthony Achilonu, Protein Structure-Function Research Unit (PSFRU), University of the Witwatersrand, South Africa 

Neglected Tropical Diseases (NTD’s) and Antimicrobial Resistance (AMR) are major challenges threatening the world’s sustainability and development efforts across the spectrum of the UN’s Sustainable Development Goals, causing millions of deaths each year1. My name is Dr Ikechukwu Achilonu and I work in the PSFRU at the University of the Witwatersrand, in South Africa, where I focus on rational design and discovery of new generation anthelminthic and anti-bacterial drug targets to tackle NTD’s and Nosocomial infections by ESKAPE2 pathogens.  

Dr Ikechukwu Anthony Achilonu, Protein Structure-Function Research Unit (PSFRU), School of Molecular and Cell Biology,
Faculty of Science, University of the Witwatersrand. ©Diamond Light Source 

To design inhibitors to serve as potential drugs against these pathogens, high-resolution 3D protein structures are needed to test the ability of certain drugs to interact with and inhibit the properties of the proteins being studied. This is made possible with the GCRF START grant which provides us with access to state-of-the-art synchrotron techniques, equipment and expertise at the UK’s world class national synchrotron, Diamond Light Source (Diamond), enabling the X-ray diffraction from protein crystals generated in our research. The GCRF START grant is currently our only conduit to the UK to enable us to conduct these ultra-high-quality experiments.  

My recent research success is solving the Schistosomiasis joponicum Glutathione S-transferase (GST) enzyme has been recently published and is a tribute to the value of collaborating with Diamond and GCRF START. The solved structure is among the crystal structures with the highest resolution in the global protein database for this enzyme with two different ligands. Of the authors, Dr Ramesh Pandian is a GCRF START postdoctoral fellow (Host: Prof. Yasian Sayed), and Dr Sylvia Fanucchi, Prof. Yasien Sayed, Prof. Heine W. Dirr, and I – Dr Ikechukwu Achilonu – are collaborators on the GCRF START grant.

The resulting publication emphasises the need to exploit the unique structural diversity between Schistosoma GST and other human GSTs for a rational approach to design new generation anthelminthics. This research, outlined later in this article, will pave the way for the next exciting step in the structural validation of druggable targets for this debilitating and life-threatening Schistosomiasis (Bilharzia), a NTD which infects large numbers of people across several countries each year4.  

Aerial view of Diamond Light Source, which is the UK’s national synchrotron. ©Diamond Light Source 2020

The extent of the challenge: NTD’s and ESKAPE pathogens 

AMR is described by the World Health Organization5 as “one of the biggest threats to global health, food security, and development today…compromising our ability to treat infectious diseases, as well as undermining many other advances in health and medicine.  

NTD’s are a diverse group of communicable diseases caused by a variety of pathogens prevalent in tropical and subtropical conditions, such as viruses, bacteria, protozoa and parasitic worms (helminths). More than a billion people in over 149 countries are impacted by NTD’s with huge costs to developing economies of billions of US dollars every year. My research on NTD’s focuses on Schistosoma and Wuchereria (Wuchereria is a human parasitic worm (Filariworm) that is the major cause of lymphatic filariasis) – both of which display AMR. Populations living in poverty, without adequate sanitation and in close contact with infectious vectors and domestic animals and livestock, are those worst affected by NTD’s6.  

The six nosocomial ESKAPE pathogens that exhibit multidrug resistance and virulence are often acquired in health care settings like hospitals (Health Care Acquired Infection/HAI) and feature on the World Health Organization’s ‘Priority Pathogens list’7. The persistent use of antibiotics has provoked the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria, which can make even the most effective drugs ineffective. These bacteria have built-in abilities to find new ways to become resistant and can pass on genetic materials which enable other bacteria to become drug resistant. Like NTD’s, this situation is made worse by limited or non-existent infection prevention and control (IPC) programmes, combined with an inadequate water supply, poor sanitation, and a weak hygiene infrastructure in health facilities, which make the burden of HAI several fold higher in low- and middle-income countries than in high-income ones.  

World class results! Solving the Schistosomiasis japonicum GST enzyme  

Over the past three years, I have been working on the Schistosoma GST enzyme, which is a protein that is involved in detoxification of foreign molecules in the Schistosome parasite that causes the disease Schistosomiasis/Bilharzia. This enzyme very critical to the survival of the parasite. If the enzyme is not functioning properly, the parasite cannot move from egg stage to lava stage. Therefore, if the enzyme could be effectively inhibited, one would have a drug against the parasite. 

In my research, I used a molecule which is a natural product found in pomegranate juice called Ellagic Acid. One of my MSc. students, Ms Blessing Akumadu, carried out the study I had conceptualised, showing that this molecule can inhibit the activity of the Schistosoma GST by 66%. The concept was based on an earlier study that ellagic acid and its derivative inhibits Plasmodium GST (duly acknowledged in the forthcoming paper).   To be more confident about making extrapolations from the structure of the Schistosoma japonicum GST enzyme, we needed to see that our wet lab results had meaning in 3D high resolution. Using the I038 and I04 beamlines at Diamond, we were able to solve the structure of the 26 kilodalton GST at 1.53 Å resolution and were amazed when we saw the results! We had achieved the highest resolution of any of the several variants of the Schistosoma japonicum GST in the global protein database!   

What we observed is the Schistosoma 26 kilodalton GST enzyme (Sj26GST), which is a dimeric protein (it has two similar subunits joined together) with an interface where the subunits are joined. The unique thing about this protein is that the dimer interface can be exploited for drug interaction, unlike other GST’s in humans. The results have enabled us to see where the Ellagic Acid molecule (the potential drug) can bind to the protein. In this parasite we only have two classes of GSTs (in humans there are several) – the 26 kilodalton (kDa) GST and the 28 kDa GST. Having studied the 26 kDa GST, we are now currently working on the 28 kDa GST across the three species of the Schistosoma parasite that infect humans (mansoni, haematobium and japonicum) to see if the Ellagic Acid molecule can also inhibit the 28 kDa GST of the Schistosoma parasite.  

The implications of these results mean we can go on and redesign this potential drug molecule because we can see the type of interaction this drug is making with the protein in the 3D images. The more the drug interacts, the stronger the binding. Therefore, we can start to redesign this drug for better binding so that the binding and the efficiency is improved beyond the 66% to maybe 75% or even to 99 % in order to completely shut down the activity of the enzyme at a very low amounts of the drug. Without the high-resolution 3D crystal structure solved using Diamond, we could not do this. We can now exploit this interaction by adding a bond here or a functional group there to improve the strength of the interaction and potency of the drug, and produce new high resolution structures until we have a drug that shuts down the enzyme and produces a new drug target to which the Schistosoma parasite is not resistant to. 

The benefit of studying multiple NTD and ESKAPE pathogens simultaneously – the case of nicotinamide adenine transferase enzyme in klebsiella pneumonia and Enterococcus faecium. 

In my experience, the techniques one applies to studying one protein can also be applied to the others, which is why I study a range of druggable proteins from several human NTD and ESKAPE pathogens simultaneously. For example, my students and I are examining the nicotinate-nucleotide adenylyltransferaseenzyme which is important in the redox metabolism in both klebsiella pneumonia and Enterococcus faeciumKlebsiella pneumonia is a Gram-negative bacterium resistant to multiple drugs and increasingly resistant to most available antibiotics (it has a thin cell wall surrounded by a protective cell membrane which makes it harder for antibodies to penetrate). In health care settings, patients who require devices like ventilators or intravenous catheters, and patients who are taking long courses of certain antibiotics are most at risk for Klebsiella infections.   

The Gram-positive Enterococcus faecium is a human pathogen that causes nosocomial bacteremia, surgical wound infection, endocarditis, and urinary tract infections (it has thick cell walls but no outer membrane). Studying both simultaneously has led to us noticing that the Gram-negative function of the nicotinamide adenine transferase enzyme performs differently from the Gram-positive function of the same enzyme which is exciting because this enables us to infer structural and functional differences between this enzymes from different bacteria that are supposed to perform similar activities in these bacterial pathogens. This has serious implication in the design of a broad-spectrum antibiotics that will target this enzyme in different organisms.  

The process of solving protein structures – systematic integration of multi-facted ‘new generation’ theoretical and empirical studies 

The approaches we use in the research outlined above are multi-faceted and multi-disciplinary involving the integration of sophisticated ‘new generation’ theoretical and empirical studies and, as already indicated, a range of cutting-edge equipment. To design drugs against disease pathogens, the three-dimensional architecture of the druggable protein is needed. Druggable proteins are proteins that are key to the survival of the pathogen which enables it to replicate inside the human host, either intracellularly or extracellularly. In the case of bacteria, for example, they need to manufacture their own energy and without the enzyme (protein) that makes this possible, the bacteria would not survive. Therefore, if we know the protein’s DNA sequence, we should be able to predict that we can synthesis this protein and develop the druggable target.  

To elucidate the crystal structure of these druggable proteins it is possible to use home X-ray sources. Ultimately, however, there is a major advantage of using a powerful synchrotron light source like Diamond over most home X-ray sources because the amount of inferences made from a protein crystal with resolutions in the range of 1.3 – 1.7 Å is unprecedented. Such crystals can be further studied using computational algorithms and can also serve as models for replacement when solving the crystal structure of a protein that is very similar in structure. 

In order to generate the recombinant proteins that are druggable targets, we need large amounts of these proteins. For this we use E. coli bacteria like a factory to synthesize our target proteins and carry out a rigorous ‘quality assurance’ process to ensure the proteins we make are pure, stable and homogenous for successful crystallisation experiments.  If the protein is an enzyme, it should be able to catalyze the same function it does naturally in an organism (in vivo), inside a test tube (in vitro), which means that whatever we send off to Diamond for X-ray diffraction experiments, we need to be sure that the biological function is intact. To do this, the proteins are assayed (measured) for catalytic activity in vitro (if they are enzymes) and are further characterised using several biophysical techniques to test their structure and the function to the point we are satisfied that the proteins are active, properly folded and can perform the function they are supposed to perform properly (biological activity is maintained).  

We purify the proteins to homogeneity using an ensemble of protein chromatography techniques. For ‘quality assurance’, we check the secondary structure content (Far-UV circular dichroism), the tertiary structure (intrinsic and extrinsic fluorescence spectroscopy); and the quaternary structure (using size exclusion HPLC). Every physical interaction between two biomolecules generates heat or absorbs heat as part of the function of the protein to interact with another molecule (molecular recognition). Therefore, to estimate the thermodynamics of protein-ligand interaction (interaction ability), we use isothermal titration calorimetry.  

Once we are satisfied that the proteins are of good quality and quantity, we begin protein crystallisation. I like to use the analogy that structural biologists are akin to photographers and using X-ray diffraction is like taking a snapshot of the protein which has been forced to assemble as repeating units in a crystalline form. There are several approaches we use. We can either conduct crystallisation manually, or we can do it robotically using our in-house Douglas Instrument Oryx 8 crystallography robot for high throughput protein crystallisation.  

To solve the protein structure – sometimes in complex with potential drugs – X-ray light is invaluable. However, X-rays have very short wavelengths and if you keep bombarding a protein crystal with X-rays, it will heat it up and gradually fall apart because of X-ray damage. By changing the protein from solution to a crystal, and then embedding it in a liquid able to resist the intense bombardment of X-ray (cryo-protection) by freezing it at the temperature of liquid nitrogen (-196 degrees Celsius), the sample stays stable and the X-rays will last 100x longer in the X-ray beam! Our crystal samples are therefore sent to Diamond in special containers which keep the samples crystals frozen. GCRF START makes it very easy to ship our crystals to the UK without lots of red tape. We use DHL and we have an easy system which means it only takes a few days to get to Diamond ready for the experiments; the GCRF START grant helps this process at no cost to us.  

We can use an X-ray diffractometer such as a small in-house diffractometer to solve the protein structure, or the ultimate technique for X-ray crystallography – a massive synchrotron like Diamond! Diamond’s I03 and I04 beamlines are used to fire X-rays onto our crystal samples. The X-ray will be scattered based on the structure of the protein/molecule (X-ray diffraction). Then, using mathematical modelling and other techniques, the 3D structure of the protein is resolved, either on its own or in a complex with a drug or other biomolecules such as DNA.   

My theoretical (computational) studies involve several molecular modelling techniques to model the atomistic behaviour of our proteins of interest, especially how the dynamics of these proteins are altered upon drug binding. We also screen various in silico (virtual) libraries (including PubChem, ZINC and Drug Bank databases) for potential hits that we further extend to the theoretical studies, including high-throughput virtual screening, induced-fit ligand docking, molecular dynamic simulation and free binding energy calculations. 

Sometimes, however, the process provides a few surprises, which was the case in our journey to solving the structure of the Schistosoma japonicum GST enzymeWe usually send our crystals to Diamond at the same time as the other groups in South Africa because we have the same slots for data collection. Two weeks before we were supposed to ship our crystals, we found that none were ready yet to ship.  This wasn’t good. Therefore, to hurry things along, I asked one of my MSc students, Jessica Olfsen, who knows the crystallization robot very well to generate the crystals we needed within one week. This she did, but they were very ugly looking crystals! I did not think we would get any data out of these crystals but at least we had crystals to send. I so doubted their usefulness that when I collected the data, I didn’t even want to look at it, let alone solve the protein structure. In my disappointment I asked one of our new post-docs (Dr Pandian) who needed a task to look at it. The resulting 3D high resolution structure blew him away! When we both looked at it, we saw just how beautiful it was. I had written the paper already, but it had needed more experimental data for publishing so now I had it! The structure was solved at unprecedented resolution and the paper was accepted for publishing. 

As a structural biologist, drug discovery and protein biochemistry are completely intertwined and multidisciplinary in the sense that I cannot do without a synthetic chemist (organic chemist). These are the people who will synthesize the drug. For example, once we add the extra functional groups, it will improve the binding capacity of the drug. The synthetic chemist will then go into the laboratory, resynthesize the drug before I will return to my lab and see if the inhibition improves. When an improvement in the inhibition is apparent, we go through the whole process again: recrystallize the protein with the proposed drug, and then use Diamond again to produce another high-resolution 3D structure of the re-modelled drug molecule and protein interaction.  

“This whole drug discovery process can take some time, so it is vital that we have access to world class synchrotron facility like Diamond for the lifetime of this research we until we have the results needed to tackle the NTD’s and ESKAPE pathogens which impact countless lives.” 

Dr Ikechukwu Anthony Achilonu, Protein Structure-Function Research Unit (PSFRU), University of the Witwatersrand, South Africa 

Read more here about the UN’s Sustainable Development Goals. 

More about Dr Achilonu 

Dr Ikechukwu Anthony Achilonu is Senior Researcher and the Interim South African Research Chair (NRF/SARChI) in Protein Biochemistry and Structural Biology at the Protein Structure-Function Research Unit (PSFRU), School of Molecular and Cell Biology, Faculty of Science, University of the Witwatersrand in South Africa.  

Publications: https://pubmed.ncbi.nlm.nih.gov/?term=Achilonu+I&cauthor_id=30183110 


[1] Transforming our world: the 2030 Agenda for Sustainable Development – A/RES/70/1. New York, USA: United Nations; 2015.

[2] ESKAPE is an acronym for the group of bacteria, encompassing both Gram-positive and Gram-negative species, made up of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. 

[3] Authors: Blessing O. Akumadu, Ramesh Pandian, Jessica Olfsen, Roland Worth, Monare Thulo, Tshireletso Mentor, Sylvia Fanucchi, Yasien Sayed, Heini W. Dirr, Ikechukwu Achilonu. Title: Molecular basis of inhibition of Schistosoma japonicum glutathione transferase by ellagic acid: insights into biophysical and structural studies. Publication: Molecular and Biochemical Parasitology (2020);Elsevier. https://doi.org/10.1016/j.molbiopara.2020.111319

[4] https://www.who.int/news-room/fact-sheets/detail/schistosomiasis

[5] World Health Organization Global action plan on AntiMicrobial Resistance (AMR). Geneva: World Health Organization; 2015.  https://www.who.int/antimicrobial-resistance/global-action-plan/en/

[6] https://www.who.int/neglected_diseases/resources/9789241515450/en/

[7] https://www.who.int/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed

SA women take the lead in structural biology

A team of researchers from the University of Cape Town (UCT) and University of the Free State (UFS) has achieved a remarkable feat in the field of structural biology by determining the structure of an enzyme that could be a key component in producing valuable commodity chemicals in greener, sustainable processes.

Known as cytochrome P450 reductase (CPR), the enzyme has received much attention – not only for its ability to perform difficult chemistry, but also for its role as drug target.

“The task was enormous,” said team member Naadia van der Bergh, a PhD student in UCT’s Centre for Bioprocess Engineering Research (CeBER). “CPR is a massive enzyme. It contains 679 amino acids and there were two molecules in the asymmetric unit. Added to that, our initial structure was determined and solved on the basis of a low-resolution map. Interpreting this structure was a truly gruelling effort.”

The results of the research were published on 27 December 2019 in the journal Scientific Reports (9:20088) by the Nature Publishing Group.

International exposure

With support from the Global Challenges Research Fund’s (GCRF) Synchrotron Techniques for African Research and Technology (START) programme, the team was given the opportunity to conduct parts of their research at the Diamond Light Source synchrotron in Oxfordshire in the United Kingdom (UK)

Read more on the University of Cape Town News.

Image: Ana Ebrecht (left) and Naadia van der Bergh are part of a team of researchers from UCT and UFS that achieved a remarkable feat in the field of structural biology.

Double first! First synchrotron user from the University of Zululand solves partial structure of the Schistosomiasis (Bilharzia) G4LZI3 universal stress protein

In a ‘double first’, Dr Priscilla Masamba, has become the first University of Zululand student to use the UK’s National Synchrotron Light Source, Diamond, and solve the partial structure of a protein from Schistosoma mansoni. With access to the synchrotron made possible by GCRF START, Priscilla employed sophisticated robotic instruments and macromolecular X-ray crystallography techniques remotely from South Africa to solve the partial structure of the G4LZI3 universal stress protein – a protein regarded as a target for novel medicines for treating the disease Schistosomiasis. The experiments took place in February 2020, using the Diamond’s I04-1 beamline.

Dr Priscilla Masamba in the laboratory at the University of Cape Town.
Photo credit Rebekka Stredwick. ©Diamond Light Source

Schistosomiasis is an acute and chronic disease caused by parasitic worms (schistosomes) endemic in more than 78 countries with an estimated 4 million people infected in South Africa alone. The disease requires an intermediate host, the freshwater snail Bulinus africanus, and occurs most often in rural areas where people become infected during routine agricultural, domestic, occupational, and recreational activities which expose them to infested water.

Only one drug, Praziquantel, is available to treat Schistosomiasis leaving people vulnerable to schistosome resistance and this treatment is only partially effective in treating adults.  The aim of Priscilla ’s research is therefore to generate insights for the design of alternative treatment regimen targeting specific stages during the developmental cycle of the schistosome.

Describing the experiments at Diamond as “close to a cool sci-fi movie,” Dr Masamba was able to control the sophisticated instruments on I04-1 beamline and collect data in real time from the University of Cape Town’s (UCT’s) Aaron Klug Centre for Imaging and Analysis – established as a GCRF START Centre of Excellence for structural biology research.

“Remote data collection at Diamond was so exciting!” Dr Masamba explains, “I could literally control and see a robot that was thousands of miles away on the other side of the world, mount a microscopic crystal (sample) within the firing line of a powerful X-ray beam, and determine the amount of energies released by light emitted from the sample caused by incident X-ray beams, and all of this while working from the laboratory in Cape Town. I didn’t need to get in a plane to achieve the one of the most imperative steps in the crystallography process! The whole experience provided me with rare exposure to the world of X-ray crystallography, impacting my view of science in a spectacular way.”

Proteins are thermodynamically and kinetically responsible for all biochemical processes that occur, and are therefore responsible for coordinating, regulating and dictating numerous metabolic functions. Exposure of the Schistosome parasite to extreme conditions during its developmental stage triggers the expression of heat shock proteins and universal stress proteins, of which the G4LZI3 USP has been identified as a potential druggable target for the development of alternative treatments (schistosomicides). Techniques like X-ray crystallography can provide insight, not only into the composition of these biomolecules, but also into their various interactions with other compounds and their roles in biological mechanisms, an imperative foundation for rational drug design and development.

Before the experiments took place, diffraction of the crystals was first checked at UCT using a diffractometer. Crystals from these conditions were then flash-frozen in liquid nitrogen and shipped to the Diamond synchrotron to be used as samples.

The BART robot and sample holder on beamline I04-1. The drum (Dewar) contains liquid nitrogen, and space for 37 pucks, each containing 16 pins, so 592 samples. These pins and pucks are shipped in a Dewar from South Africa. The robotic arm is grey and is shown ready to pick up the next sample. When it selects the next sample, this is placed onto the goniometer, which holds the sample and rotates it for data collection.
©Diamond Light Source.
The goniometer on beamline I04-1 holds the microscopic crystal on a pin with the sample on the end of it which rotates in the firing line of the powerful X-ray beam.
©Diamond Light Source.

The solved structure of the S. mansoni G4LZI3 is a success story for the University of Zululand, a small resource-constrained university in the rural part of KwaZulu-Natal Province of South Africa. The University of Zululand lacked the resources required for Dr Masamba to achieve all her objectives for her PhD, which meant the collaboration through START in order to carry out the experiments needed was imperative both professionally and personally.

Priscilla is thankful for the guidance and mentoring from her PhD supervisor, Professor Abidemi Paul Kappo, who heads up the Biotechnology and Structural Biology (BSB) Research Group in the Department of Biochemistry and Microbiology at the University of Zululand, and from START Principal Investigator, Professor Trevor Sewell, of UCT’s Aaron Klug Centre for Imaging and Analysis, both of whom helped Priscilla overcome various challenges.

“I have been able to learn and cultivate scarce, critical and sought-after skills here in Africa in the fields of bioinformatics and drug discovery, molecular biology and especially, structural biology,” says Dr Masamba. “These include gene cloning, recombinant protein expression and purification, as well as characterisation of proteins. This has not been an easy task because I am from an underrepresented group in science as a black female and study at a historically-disadvantaged and resource-constrained institution.”

Professor Abidemi Paul Kappo, (left) head of the Biotechnology and Structural Biology (BSB) Research Group in the Department of Biochemistry and Microbiology at the University of Zululand, and START Principal Investigator, Professor Trevor Sewell (right), from the University of Cape Town’s Aaron Klug Centre for Imaging and Analysis.
©Diamond Light Source.

An important objective of the START programme is to increase the number of structural biologists in similar less developed universities in South Africa and across the continent. This can present complex challenges, not least because many students are ill-equipped for work in the field of structural biology.

“A key concept behind the creation of the START Centre of Excellence at UCT’s Aaron Klug Centre for Imaging and Analysis, for example, is to provide the necessary infrastructure to enable senior students and staff at South Africa’s historically disadvantaged universities to access both the human and material resources necessary to overcome the difficulties and determine protein structures,” Professor Sewell says. “We count the collaboration with Professor Paul Abidemi Kappo and Dr Masamba as a major success in this respect.”

This collaboration between Prof. Kappo and Prof. Sewell was enabled by GCRF START with Prof. Sewell providing the technological resource for the G4LZI3 structural biology project, as well as the linkage to Diamond.

“Above all, Professor Sewell’s enthusiasm to train and develop a “critical mass” of students in Structural Biology is second to none,” Prof. Kappo says. “This has been a joint effort and a model of national and international collaboration. In addition to the technological resources through UCT and linkage with Diamond in the UK, funding for this project was provided by the National Research Foundation (NRF) of South Africa through a doctoral bursary awarded Dr Masamba. It is expected that structure-guided drug discovery for schistosomiasis will be the concluding part the project.”

Dr Masamba and Prof. Trevor Sewell with colleagues collaborating with GCRF START at the Aaron Klug Centre for Imaging and Analysis at the University of Cape Town. In the picture from left to right: Dr Priscilla Masamba, Dr Jeremy Woodward, Melissa Marx, Dr Mulelu, Dr Philip Venter, Dr Lizelle Lubbe, Professor Trevor Sewell
Photo Credit: Rebekka Stredwick. ©Diamond Light Source.

About Dr Priscilla Masamba

Born to Congolese parents in the DR Congo, Dr Masamba lived in the UK and Zimbabwe as a child, before moving to South Africa where she matriculated and studied for her first degree in Biological Sciences at Walter Sisulu University, Mthatha. Thereafter, Priscilla joined the Biotechnology and Structural Biology (BSB) Research Group in the Department of Biochemistry and Microbiology at the University of Zululand headed by Prof Abidemi Paul Kappo and registered under his tutelage for a BSc (Hons) degree, followed by an MSc and later a PhD in Biochemistry. Priscilla’s desire is to continue in the path of macromolecular X-ray crystallography of proteins through the NRF Postdoctoral Fellowship in Structural Biology at the University of Johannesburg.


Dr Priscilla Masamba extends a special thanks to Dr Brandon Weber (UCT), Dr Phillip Venter (UCT), Kaylene Baron (UCT), and Ndibonani Qokoyi (University of Zululand) who were involved in different ways in the production, purification and crystallisation of the G4LZI3 protein, as well as in data collection.

Structural biology – Improvements in health

The need for health improvement on the African continent continues to be a pressing issue, and START’s emphasis will be on diseases such as HIV-AIDS, malaria, tuberculosis, and African horse sickness that are devastating to human and animal populations. The structural biology strand of START research will support scientists in finding and developing cures by researching and understanding the fundamental molecular structure of certain diseases. Prof. Trevor Sewell from the University of Cape Town explains:

“START will allow us to understand drug targets and cure African diseases. We will establish a collaborating network of seven South African institutions (the Universities of Pretoria, Witwatersrand, North West, Free State, Stellenbosch, Cape Town and the National Institute for Communicable Diseases) that will enable young researchers to boost medical and veterinary research”.

Prof. Trevor Sewell, University of Cape Town

A START project at University of Cape Town led by co-investigator Prof Edward D Sturrock

ACE in complex with the clinically used antihypertensive drug, lisinopril (black sticks; PDB ID: 1O86)

Structural biology of angiotensin converting enzyme and related metalloproteases

Enzymes play important roles in a variety of biological processes in the human body. Angiotensin converting enzyme (ACE) for example, is a metalloprotease which regulates blood pressure and is also involved in scar tissue development (fibrosis). Conditions such as diabetes and tuberculosis can lead to excessive scar tissue formation, which ultimately stops proper organ function. Currently, there is no specific treatment for fibrosis and affected individuals have an average survival period of 2-4 years. Hypertension, on the other hand, is a major risk factor for cardiovascular disease and stroke, which accounted for 15.2 million global deaths in 2016. Our research group, led by Prof Edward Sturrock, is based in the Department of Integrative Biomedical Sciences at the University of Cape Town and has a long-standing interest in ACE and related zinc metalloproteases.

Although ACE inhibitors reduce fibrosis and are widely used for treating hypertension, certain patients experience the life-threatening side-effect of severe swelling below the skin surface of the throat and tongue. With the resources provided by START, we aim to design compounds devoid of this side-effect. Detailed structures of ACE will be solved using X-ray crystallography and cryo-electron microscopy to improve our understanding of how ACE functions and enable the design of antifibrotic and antihypertensive drugs.

START Collaborators – research projects

For information on projects please click on the names below

Stellenbosch University: Professor Erick Strauss and Anton Hamann, post-doc 

Cape Town University, Lauren Arendse

The START of great things!

Visualising the structure of an intact helical filament at close-to-atomic resolution for the first time

“We are seeing critical scientific discoveries and the emergence of a new generation of experts that have resulted directly from our training programmes in advanced methods and the use of synchrotron facilities and tools.” Dr Gwyndaf Evans, START Principal Life Sciences Principal Investigator and Principal beamline scientist on Diamond’s VMXm beamline.

A seminal work of Dr Jeremy Woodward, Dr Andani Mulelu and Angela M.Kirykowicz from the University of Cape Town (UCT), South Africa, has provided novel and exciting insights into the structure and inner workings of nitrilase enzymes with the potential to address key health, food security and environmental challenges within Africa and beyond.

The results were published on the 17 July 2019 in Nature Communications Biology 2:260 (2019)4 and made possible through the UK’s national synchrotron light source, Diamond Light Source, which has integrated facilities for life sciences users as a ‘one stop shop’ for structural biology.

Dr Mulelu and Dr Woodward in front of the cryo-electron microscope
at the University of Cape Town.
Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.
The first high resolution visualisation of a Cryo-EM6 protein structure in Africa

Nitrilases are a class of plant enzymes that play an important role in the synthesis of a broad range of chemicals. Although have specificity for a small range of substrates they have a large potential to create products of biotechnological significance.

Building on more than a decade of structural biology research, and exploiting knowledge sharing and synchrotron access opportunities through the Global Challenges Research Fund’s GCRF START Programme, Dr Woodward, Dr Mulelu and Angela Kirykowicz were able to visualise the structure of an intact helical filament at close-to-atomic resolution for the first time – the first high resolution visualisation of a Cryo-EM6 protein structure ever to be produced in Africa (Fig 1).

The scientists achieved this at the UK’s national Electron Bio-Imaging Centre (eBIC), using Cryo-Electron Microscopy, on the Titan Krios III (Beamline M06) – one of Diamond’s ‘super microscopes’ – to observe how the maximum size of a bound substrate is limited by a loop which shifts with helical twist after mutating a single amino acid.

Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.

Observing the ‘loop’, ‘lock’ and ‘lid’: Dr Woodward describes their observations in terms of a ‘loop’, ‘lock’ and ‘lid’: the size of the binding pocket seems to be limited by a ‘lid’, which prevents long substrates from being converted. This lid is formed by a ‘loop’ which is held in position by electrostatic force. A single amino acid is responsible for maintaining this interaction – this is referred to as the ‘lock’. Other amino acids within the binding pocket interact with various chemical groups of the substrate and either allow it to bind or prevent it from binding. Two regions in particular are important for this effect. The three amino acids are: “the lock”, which defines the overall size of the substrate that can bind (either by tightening or loosening the lock by altering its chemical properties); and two others within the binding pocket, which interact with the substrate directly.

The insights gained allowed the team to semi-rationally design a new mutant nitrilase enzyme that produces biotechnologically interesting products, whether pharmaceuticals, fine chemicals or even food.

“We did this by identifying ‘hot spot’ amino acids for directed evolution and selecting them by coupling the survival of bacteria to the successful conversion of a library of substrates,” explains Dr Woodward, who had identified the most important of these residues ten years ago.

“Previously, at low low-resolution, we had seen large-scale changes,” Dr Mulelu adds “but we needed to answer the question: “How did this work? How did these translate into differences that could account for the ability of these enzymes to distinguish between substrates that differ by only one carbon atom? The Titan Krios III (beamline M06) housed at eBIC enabled us to answer our questions and see the enzyme at atomic scale resolution and to achieve these wonderful results.”

Dr Mulelu setting up the Vitrobot ® for preparing grids for cryo-Electron Microscopy. Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.
Designer nitrilase enzymes

These results pave the way for further exciting research opportunities. Going forward, Dr Woodward’s ultimate aim is to produce a ‘catalogue’ of ‘designer’ nitrilases’ in a quest to find solutions through biotechnology which could lead to sustainable transformations in the lives and livelihoods of populations across Africa.

“My vision is to create a ‘catalogue’ of ‘designer nitrilases’ for any substrate by making appropriate changes to the helical twist as well as the binding pocket,” Dr Woodward says. “To achieve this, we would like to visualise a collection of key nitrilases with a range of different helical states (and substrate specificities). We are currently using computer modelling to predict binding energies and correlate these with known substrate specificities of various binding pocket mutants we have.”

Meeting the global challenges

The results achieved by the UCT team could play an important part in providing sustainable solutions in the future to meet the UN’s Sustainable Development Goals, from novel drug design and manufacturing to tackle communicable and non-communicable diseases, to pioneering ‘green’ biotechnology options for agricultural food security, industrial and mining waste treatment using enzyme-catalysed products.

Medical drug discovery and manufacturing

Dr Woodward is convinced that investments in new drug manufacturing methods may have a bigger impact on health on the African continent than the development of entirely new drugs,

”Africa has the worst burden of life-threatening communicable diseases in the world,” explains Dr Woodward. “According to the UN, 1.6 million people died from a combination of preventable and treatable diseases in 2015 – malaria, tuberculosis and HIV-related disease. A big problem in Africa, however, is access to medicines and not necessarily the development of new medicines. Part of the problem is that only 2% of the medicines used in Africa are manufactured in Africa, which has implications for the cost and accessibility of medicines.”

This is where nitrilase enzymes could provide solutions. Nitrilase enzymes are attractive biocatalysts for the synthesis of amides and carboxylic acids for use in the manufacture of drugs for major life-threatening communicable diseases, such as malaria, tuberculosis and HIV-related disease.

Drug discovery and testing for these diseases would also benefit from the economies of scale of an ‘off the shelf’ catalogue of nitrilases manufactured in Africa, making drugs for these diseases much more accessible and affordable.

“The ability to manipulate how nitrilases work,” Dr Mulelu points out, “could enable more cost-effective manufacturing of drugs for such diseases, leading to cheaper and more accessible medicines and this is welcome news for us in Africa and in other developing countries.

In addition, manufacturing locally has a variety of advantages including lowering costs of transport and improving local economic development. Nitrilases can offer ‘green’ alternatives because the reaction occurs at (close to) room temperature, neutral pH and atmospheric pressures so require less energy to produce. They are also very specific and so produce less waste.”

‘Green biotechnologies’ for agriculture and waste treatment

Looking beyond pharmaceuticals, nitrilases have a host of potential ‘green technology’ solutions to offer for cleaning up environmental pollution. One example is the breaking down of cyanide, which could revolutionise the way we clean hazardous mining dumps, reduce water pollution and improvements in the treatment of industrial waste and green manufacturing using enzyme-catalysed products.

“The nitrilase 4 mutant we have produced (described in the latest paper) can break down cyanide, forming products that can be further broken down by bacteria,” Dr Woodward explains. “This also applies to farming and food security. These compounds release cyanide and animals, especially ruminants, can suffer cyanide poisoning as a result but nitrilases with specificity towards cyanide could be used in a genetically modified strain of sorghum, which could break down the cyanide and make it safer.”

Developing a new generation of research leaders

A significant focus of the START programme focuses on capacity building and investing in the development of existing and future science talent across the continent, as well as funding Post-Doctoral Research Assistants and Fellows, and resourcing laboratories. For Dr Andani Mulelu, the impact of being a ‘Synchrotron Techniques for African Research and Technology (START) Postdoctoral Research Fellow’ has been significant, particularly in achieving results,

“During my honours year I became fascinated by the structure of helical nitrilases, especially those that detoxified cyanide, and I joined the group of Professor Trevor Sewell for my Masters and later my PhD. Working with Dr Jeremy Woodward and GCRF START enabled me to finally to realise my dream of visualising a nitrilase at atomic resolution and to solve the mystery of substrate selectivity in these enzymes.”

Dr Mulelu was subsequently offered a job as a research scientist at the H3D Drug Discovery and Development Centre (UCT) working on Malaria and Tuberculosis target-based drug discovery programmes, a move he attributes to the experience he gained as a START-funded post-doc.

Dr Andani Mulelu
Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.

Ms Kirykowicz was a Masters student at the time the team achieved the successful results described in the Nature paper. Interested in applying the directed evolution techniques from her Honours year, Ms Kirykowicz’s role in the team involved working on nitrilase specificity, which gave her the skills she needed her future studies,

“The START-funded project gave me a good understanding of the methods needed to produce a well-sampled mutant library. I liked structural biology and ended up completing my Master’s on solving Mycobacterial protein complexes with Dr. Woodward. I am currently doing a PhD at the University of Cambridge in the UK working on solving the cryo-EM structure of a protein toxin transporter.”

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