CCP4 Crystallographic School South Africa 2021

Data Collection to Structure Refinement and BeyondDigital Conference

The first CCP4 Crystallographic School South Africa took place on the 22 February – 5 March 2021 hosted online due to the COVID-19 pandemic.  More than 90 attendees – including 39 students and postdoctoral researchers represented by 14 different nationalities – participated in the workshop, which covered fundamental topics relating to theoretical concepts and practical approaches in protein structure solution by X-ray crystallography.

The event involved both newly emerging scientists as well as seasoned experts from across Africa, the UK, Europe, the USA, and Australia and was supported and co-hosted by the GCRF START grant, the UK’s national synchrotron – Diamond Light Source (Diamond), the Collaborative Computational Project Number 4 (CCP4), and the University of Cape Town (UCT). The workshop consisted of informal social events, formal lectures, question and answer sessions, one-on-one tutorials on data processing, case studies, and data collection where students collected data remotely from Diamond or provided their own data.

Aerial Coastal view of Cape Town, South Africa. ©CCP4_mx Crystallographic School, South Africa

Describing the impact on the small but growing structural biology community in Africa, workshop co-organiser, Professor Trevor Sewell from UCT’s Aaron Klug Centre for Imaging and Analysis, said,

The value of knowing a protein structure is widely appreciated by the scientific community but the knowledge and experience of how protein structures are determined is rare in Africa. The Covid-19 pandemic has shown us that we remain ignorant of this key area of science that has already led to successful vaccines and may lead to valuable drugs at our peril. The pandemic has also focused our minds on finding new ways of working and this has enabled us to hold this extraordinary workshop remotely. This has enabled African students to engage with the world’s best without the need to travel.”

“The success of the new medium was extraordinary and can potentially be extended to cover all fields. The workshop was the brainchild of Dr Carmien Tolmie from the University of the Free State here in South Africa, and it owes its success to her dedication and organizational abilities. We are grateful for the generous sponsorship from the National Research Foundation, IUPAP and the IUCr, which made this trendsetting virtual workshop possible,” Prof. Sewell added.

Three beamlines at Diamond Light Source, namely i03, i04 and i24, were dedicated to the remote data collection of students’ protein crystals, and each student was allocated to a beamline appropriate for their crystal system, as well as one-on-one assistance from beamline scientists. The workshop also had a coordinated effort involving beamline scientists from Diamond’s MX team. Diamond MX support scientists and co-hosts of the workshop – Felicity Bertram, Elliot Nelson and Marco Mazzorana – ensured the samples belonging to the workshop participants reached the correct beamline for the dedicated data collection day, in addition to organising access to the computing resources at Diamond Light Source for the data processing sessions.

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

Commenting on the importance of co-hosting the event from a South African perspective, Dr Carmien Tolmie said,

“It is an immense privilege to be part of the organization of this workshop and I am extremely grateful to the other members of the organization who worked extremely hard to make sure that this workshop was realized, despite the numerous setbacks we encountered because of the Covid-19 pandemic. Due to the high cost associated with traveling overseas to attend CCP4 workshops on other continents, this was a once-in-a-lifetime opportunity to learn from and engage with experts on crystallographic data processing for many of the participants attending this workshop. These are scarce skills, and this workshop will greatly aid in developing human capital in the country, as well as have a marked impact on advancing the projects of the participants who attended the workshop.”

For some of the students this was the first time attending a crystallographic workshop, including Taryn Adams, an MSc student recently starting a project in protein structure and function. Taryn heard about the workshop through her supervisor, Professor Yasien Sayed, head of the Protein Structure and Function Research Unit at the University of the Witwatersrand in South Africa. Taryn said,

In science, success is often achieved with significant collaboration and group learning. I am looking forward to meeting scientists who are also new to the field and those with experience who could advise me as I embark on my project.”

Taryn Adams, an MSc. student from the Protein Structure and Function Research Unit at the University of the Witwatersrand, South Africa, participating in the 1st CCP4 Crystallographic School South Africa workshop via online platform.
Photo credit: Taryn Adams. ©Diamond Light Source

Another participant, Dr Stanley Makumire, is a GCRF START funded Postdoctoral Research Fellow at the University of Cape Town. Stanley said,

“Being a novice in the field of structural biology attending the CCP4 workshop will equip me with the necessary skills for my research project which is to understand the mechanism of the amidase enzyme family. I am also really excited about the remote data collection and data processing tools available on CCP4.”

Dr Stanley Makumire, GCRF START Postdoctoral Research Fellow at the University of Cape Town, one of the participants in the 1st CCP4 Crystallographic School in South Africa. Photo credit: Stanley Makumire ©Diamond Light Source

Commenting on the impact of the GCRF START grant on capacity building in the field of structural biology, Dr Gwyndaf Evans, START co-Investigator and Principal beamline scientist on Diamond’s VMXm beamline, said,

“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 Phillip Venter at the 1st CCP4 Crystallographic School in South Africa collecting data remotely from the UK’s national synchrotron, Diamond Light Source.
Photo credit: Phillip Venter ©Diamond Light Source

New collaboration opportunities for computational insights into catalysis

A successful secondment by GCRF START computational scientist, Dr Michael Higham, has led to exciting new computational modelling collaborations involving leading catalysis institutes in South Africa and the UK.

These opportunities range from investigating adsorption induced magnetisation changes in nickel catalysts, to research into bimetallic catalysts for CO2 hydrogenation of environmental and industrial importance in the search for sustainable, clean energy sources to tackle climate change.

Dr Higham, who is a START-funded Postdoctorate Research Associate at Cardiff Catalysis Institute working with the UK’s national synchrotron light source, Diamond Light Source and the UK Catalysis Hub, spent two months from December 2019 to January 2020 at the University of Cape Town meeting researchers, undertaking initial computational work, and getting to know the projects.

Now back in the UK, Dr Higham’s aim is to provide theoretical inputs through computational modelling in order to support findings from experimental results.

One of these projects focusses on bimetallic alloy catalysts for methanol synthesis and conversion involving Dr Mohamed Fadlalla and Christopher Mullins from the University of Cape Town’s Centre of Catalysis and c*Change, South Africa’s DST-NRF Centre of Excellence in Catalysis Research.

“In early December 2019, our team from the University of Cape Town and Southampton University (UK) visited the Diamond Light Source synchrotron and used the B18 beamline to study the influence of substituents in the ferrite structure on the reduction behaviour and carbon dioxide hydrogenation reaction to valuable products (e.g. fuels),” Dr Fadlalla explains. “The next step is Michael’s computational work to help us to calculate certain insights from the results such as how the catalyst looks and how the reactant is interacting with the catalyst.”

The computational calculations will examine the catalytic product distributions which provide detailed insights into possible explanations for observed catalyst selectivity, as Dr Higham explains,

“Through modelling adsorption energies, activation energies and reaction energies we hope to shed light on what happens on the catalyst’s surface. We want to compare the observed, experimental data with the computational calculations.”

Michael Higham (L) & Mohamed (R) Fadlalla working on bimetallic alloy catalysts for methanol synthesis and conversion using computational studies.
Photo credit: Rebekka Stredwick; ©Diamond Light Source Ltd.

Another project focuses on the rationalisation of experimentally observed adsorption-induced changes in magnetisation of Ni particles. Working together with Dominic de Oliveira from UCT, Dr Higham’s intention is that the computational results, in conjunction with the experimental work, will pave the way for possible new techniques to employ magnetism to probe surface area and composition.

“The secondment was a great opportunity to meet some really enthusiastic scientists doing some excellent work on catalysis,” Dr Higham reports. “The collaborations represent not only a trajectory for progress in solving real-world energy problems but also a fundamental knowledge foundation that can inform future studies”.

“Computational experts like Michael can predict how certain catalysts perform and we can try to confirm that with our techniques which is a reciprocal learning process,” says Professor Michael Claeys, Director of c*Change. “This expertise informs us and we inform them through the results which is something a single group can’t do because very specific expertise is needed. Collaborating provides some of the most powerful learning opportunities which really shape your people.”

Such collaborations through START not only enable shared learning, they increase opportunities for publications and – in Dr Fadlalla’s words – “give Africa a bigger footprint in the wider catalyst society”, as Co-I on the START grant, Dr Peter Wells, explains,

“START is a fantastic opportunity for the UK to work in concert with our African partners to tackle shared challenges. It is clear that the excitement generated can be inspirational and helps to further integrate research partnerships.”

More about the contributors

Dr Michael Higham works with Professor Richard Catlow’s group at Cardiff University and is supported by GCRF START to provide computational insights for experimental work utilising the synchrotron facilities at Diamond Light Source Ltd. Michael’s current research project concerns Cu-based catalysts for methanol synthesis from CO2.

Dr Mohamed Fadllala is a Post Doctorate Fellow at the University of Cape Town’s Catalysis Institute in the Department of Chemical Engineering.

Dr Peter Wells is an Associate Professor and a Co-I on the START grant. He currently has a joint appointment between Diamond Light Source Ltd and the University of Southampton.

Professor Michael Claeys is the Director of DST-NRF Centre of Excellence in Catalysis, c*change, hosted by the Catalysis Institute in the Department of Chemical Engineering at the University of Cape Town, South Africa

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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