Biochemistry and X-rays, neutrons and advanced computing

“It is fascinating! I believe the GCRF START grant has laid a strong foundation for me to becoming an independent early career Structural Biologist. Now I am hands-on, a biochemist being chaperoned into the world of X-rays, electrons, neutrons, quantum mechanics and GPUs[1]

Dr Stanley Makumire, GCRF START Postdoctoral Research Fellow, University of Cape Town, South Africa.

My name is Stanley Makumire. I was born in Zimbabwe and have had a passion for Maths and Science for as long as I can remember. My desire is to gain an understanding of how disease works at the atomic level and thereby address important Sustainable Development Goals for health and wellbeing (SDG3). I had not encountered structural biology until I attended the Biophysics and Structural Biology at Synchrotrons workshop in Cape Town in early 2019 (17‐24 January), which was jointly funded by the GCRF START grant and the International Union of Pure and Applied Biophysics. At that meeting, I saw the cutting-edge work being done by people in the GCRF START programme and realised that understanding macromolecular structure was the key to understanding biochemistry, and this has inspired my research journey ever since. At the time I was completing my PhD at the University of Venda in the Limpopo province of South Africa, where none of the resources to do such work were available. Therefore, I was determined to collaborate with the GCRF START programme at the University of Cape Town (UCT).

Dr Stanley Makumire, GCRF START Postdoctoral Research Fellow, University of Cape Town, South Africa. ©Diamond Light Source

Given the disease burden in Africa, my main career goal is to eliminate or minimise disease progression using molecular and structural biology tools, with proteins as targets using small molecules designed by rational processes as drugs. This knowledge has provided insights used to design medicines and vaccines. I am motivated by the rapid advance of biophysics that we have witnessed in response to the COVID-19 pandemic. This has clearly demonstrated the power of today’s visualisation technology and the field of Structural Biology in vaccine design.

Studying the mechanisms of enzymes of the nitrilase superfamily

I was motivated to join Prof. Bryan Trevor Sewell for my Postdoctoral Fellowship, which I was awarded through the GCRF START grant in 2020. Prof. Sewell is a GCRF START Co-investigator in the Structural Biology Research Unit at the University of Cape Town.  I had applied to do postdoctoral studies on the mechanisms of enzymes of the nitrilase superfamily. These ubiquitous enzymes play a variety of roles in cellular processes, and many have found industrial roles in chemical synthesis and environmental protection. However, discovering how they work has been beset with difficulties.

It has been known for some time that three different amino acids play a pivotal role in their function: a cysteine, two glutamates and a lysine. Excellent clues have come from X-ray crystallographic studies (Fig.1), but the literature contains a multitude of different interpretations of the available evidence. The problem is that X-rays cause the cysteine to become oxidized and cannot image hydrogens (which are a key players) and electrons destroy the glutamates so that they are invisible in images obtained by electron microscopy. The tricks to circumvent these problems used by various investigators have introduced artifacts of their own and therefore a definitive mechanism has eluded humanity.

Fig.1. A glutaramide substrate bound in the active site pocket of a modified thermostable amidase as visualised using ID04-1 at the UK’s national Diamond Light Source synchrotron. The cysteine normally found at position 146 has been replaced by an alanine – thus deactivating the enzyme and enabling the bound substrate to be visualised. ©Trevor Sewell. PDB ID: 6YPA; authors: Sewell, B.T., Su, S., Venter, G., Makumire, S. (2020). The C146A variant of an amidase from Pyrococcus horikoshii with bound glutaramide. DOI: 10.2210/pdb6ypa/pdb

Overcoming the challenges with neutrons

Neutrons are known to be the least damaging of all atomic resolution imaging probes and furthermore, they enable the imaging of hydrogens (in fact, deuterium that has exchanged with the natural hydrogen). But imaging by neutron crystallography is also beset by difficulties, including the fact that the crystals required must be enormous making it necessary to prepare vast quantities of protein. To add to these difficulties, there are only six suitable neutron beams in the world, so access to an appropriate facility is very restricted. I have overcome these difficulties with the help of Zoë Fisher, who is the group Leader of the Deuteration and Macromolecular Crystallization (DEMAX) platform at the European Spallation Source, and Mathew Blakeley, instrument scientist at the Quasi-Laue diffractometer (LADI-III) at the Institut Laue-Langevin (ILL). I am in the process of collecting data and learning of the remarkable insights that can be obtained by using neutrons to study the enzyme mechanism. Determining a neutron structure of this amidase will form the basis of proposing a novel mechanism and I am excited since the preliminary results are very promising. This would be a game changer!

Opportunities, resources, and capacity building through the GCRF START grant

“GCRF START has provided me with opportunities and resources that I would never have believed were possible to access from the African continent. Participating in the START grant programme has been an extraordinary experience.”

During the first year of my GCRF START-funded Fellowship, I was able to collect, process and interpret X-ray data, process and interpret high resolution cryoEM data collected at the world-class Electron Bio-Imaging Centre  (eBIC) at the UK’s national synchotron, Diamond Light Source, thereby solving the structure of the Plasmodium falciparum glutamine synthetase (deposited as EMDB entry ID EMD-12589). I used molecular mechanics software to explore the active site of amidases and interpret quantum mechanical models of the amidase active site, thus adding substantial value to the X-ray structures. I have participated in projects related to anti-malarial drug design and had the opportunity to reinterpret the literature on amidase mechanism.

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

I have met and interacted with leading scientists through my GCRF START Fellowship who have helped me to achieve my objectives. I have also met other early career scientists involved in START projects and collaborations, and I am amazed at how far they have progressed. I am excited that, because of the GCRF START grant, I am becoming one of the very small cohort of young people in Africa that have the knowledge to contribute to the field of Structural Biology.

Commenting on Stanley’s research progress and his involvement in multiple international collaborations, Prof. Sewell said,

“Stanley has maintained spectacular productivity in spite of the challenges caused by lockdown due to the Covid-19 pandemic. He has engaged with a remarkable array of science and technology at UCT’s Aaron Klug Centre for Imaging and Analysis and has built rapidly on the work of others to bring several projects to fruition. His enthusiasm and passion have been maintained by interaction with the GCRF START team and he has leveraged this network to make contact and collaborate with neutron crystallographers at ILL and DEMAX. This clearly demonstrates the process by which capacity has been built in Africa through the GCRF START grant.”

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

[1] Graphics Processing Units