Tag Archives: structural biology

Essential metals for crystallographers: copper, gallium and a lot of gold

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As the Newcastle Structural Biology lab, prepares to enter a new era with a fantastic state-of-the-art new in house X-ray system, we asked Rick to give everyone a perspective of the last 14 years of Protein Crystallography in Newcastle on behalf of all of us – Arnaud, Martin, Jane, Paula, Bert, Owen and Jon. 

by Prof Rick Lewis

A little over 20 years after von Laue, Friedrich and Knipping collected the first X-ray diffraction experiments on copper sulphate in 1912, Bernal and Hodgkin conducted the first macromolecular crystallography experiments – on pepsin, which was first crystallised by Northrop in 1928. These, and other early pioneers (including father-and-son Nobel laureates WH and WL Bragg), established the principles by which Kendrew and Perutz solved the first 3-D protein structures, myoglobin and haemoglobin, in 1958 and 1959.

If we fast-forward more than 40 years to the RAE2001, and a stated ambition for the immediate predecessor of ICaMB was to establish a protein crystallography group in Newcastle – I think it fair to say the the University was a little slow to recognise the value of structural biology!

Steven Hardwick was so excited at the lab’s opening in 2003 that he fell asleep

Nonetheless, the Director at the time, Monica Hughes, notably aided and abetted by Steve Yeaman, Jeremy Lakey, Bernard Connolly and Harry Gilbert, coerced the powers-that-be to dig behind the sofa for some loose change and, to cut a long story short, the Newcastle Structural Biology lab (NSBL) was born in 2003.

 

 

Rick preparing to shoot some crystals in the brand new kit back in 2004

Rick preparing to shoot some crystals in the brand new kit back in 2004

Back then, there were just two prime users of the X-ray diffractometer, my group and that of Mark Banfield (who left for the John Innes in 2008). Mark’s departure opened up an opportunity for Susan Firbank to establish her own research group, which in turn meant that Arnaud Baslé replaced Susan as the X-ray facility manager. In between Susan’s sudden departure in 2011 for a new life as a pharmacist, our colleagues in NICR, Martin Noble and Jane Endicott  were recruited to NICR. Paula Salgado arrived in 2012, Bert van den Berg and Owen Davies in 2013, and Jon Marles-Wright, whose beatific face used to light up the hoardings around the INTO building site, reappeared (after a sojourn up in Edinburgh) in Biology in 2016. In 6 years, the NSBL grew from a single PI to seven, plus Arnaud. The active user group now comprises well over 30 names.

In 2015, the University announced it had set up the Research Infrastructure Fund (RIF), a £31M pot of gold to which like-minded groups could apply for funds for new equipment and other essential infrastructure. To be honest, the RIF had passed under all our radars, and we were first alerted to it by a small molecule crystallographer in Chemistry. We put together what we thought was a strong case and were very pleased to hear just before Christmas 2015 that our bid was successful. Ironically, our colleague in Chemistry’s application to the same round was, ahem, unsuccessful, but a big hand to Mike for the heads-up!

Bye bye Miss Inverted Phi*: a proper farewell to the long serving old generator (*with apologies to Don Mclean)

So a year later, after a period of testing and comparing different options and going through a remarkably painless procurement procedure, we serenaded the final shut-down of the 13-year old X-ray equipment over a glass of prosecco. Bert and Martyna both won prizes for guessing closest how many operational hours (>95,000) the old generator had clocked up. The floor in the X-ray lab was relaid (humble apologies for the noise and the smell…) and we have just installed our brand new box-of-tricks.

So what do you get these days for a little under a million pounds? Not only a state-of-the-art X-ray detector, but a revolutionary new X-ray source. Instead of firing electrons at a rotating copper drum to generate X-rays, the new X-ray source relies upon the application of an electric current across a jet of liquid gallium, to produce an X-ray beam with the best spectral characteristics and intensity in the market place. Allegedly.

Almost ready to start shooting crystals!

Arnie, our new X-ray system, almost ready to start shooting those crystals!

Moreover, we have been early adopters of this brand new technology – in fact we are the first and currently only UK group with such an instrument! Exciting times indeed.

The upshot is a 100-fold improvement in performance. Data sets that would take an entire weekend to collect can now be done in 20 minutes, about what it would have taken to collect just a single image with the old system. The final piece of the jigsaw is expected in the late spring, when a sample changer will be installed. This robotic slave will allow us to load up 48 samples into a dewar of liquid nitrogen for the programmed sample changer to load one at a time onto the X-ray machine, take test exposures, rank sample quality, and then go back and collect full data sets on the best crystals. All of this without any user intervention. Amazing. If you’d told me back in 2003 that we would have this capability in-house before both Bernard and Harry had retired, I’d have laughed out loud….

Determination is key – Prof Ramakrishnan’s Baddiley Lecture

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By Kevin Waldron.

Last week saw ICaMB host the latest in our series of Baddiley lectures, which commemorates Professor Sir James Baddiley (1918-2008). Baddiley was a distinguished Professor at Newcastle University (1954-81) and a Fellow of the Royal Society (elected 1961) who made numerous important fundamental discoveries in microbiology, not least the discovery of teichoic acids, cell wall components in Gram positive bacteria.

Jeff Errington introduces Venki Ramakrishnan to the ICaMB audience

Jeff Errington introduces Venki Ramakrishnan to the ICaMB audience

Baddiley’s work on the fundamental processes of bacteria, including the structure and function of components of the bacterial cell wall, is continued to this day in Newcastle through the work of members of ICaMB’s Centre for Bacterial Cell Biology (CBCB). Although James Baddiley died shortly after the first in ICaMB’s series of Baddiley lectures, we were delighted that the Baddiley family was again represented at this year’s lecture by James’s son, Christopher Baddiley.

This year’s guest speaker was Professor Sir Venki Ramakrishnan, distinguished research leader and Deputy Director of the Laboratory of Molecular Biology in Cambridge, Nobel laureate and newly-elected President of the Royal Society. With all of the demands on his time that come with this new role as President, the large audience that gathered on Friday afternoon were grateful that Venki was able to find time to visit Newcastle to deliver his lecture. As ever, both the lecture and the surrounding celebration was expertly organised and introduced by CBCB Director, Professor Jeff Errington.

Venki illustrates the structure of the yeast mitochondrial ribosome

Venki illustrates the structure of the yeast mitochondrial ribosome

Venki’s lecture gave a brief history of his atomic-resolution structural studies of the ribosome, the macromolecular nucleoprotein complex that converts the four-letter genetic code in nucleic acid into the twenty-letter amino acid code in proteins. He presented detailed structural models of eukaryotic ribosomes, derived from X-ray crystallography and cryo-electron microscopy data accumulated over 30 years of detailed study in his laboratory.

We asked some of ICaMB’s early career researchers to describe their impression of Venki’s lecture:

“Professor Venki Ramakrishnan was kind enough to deliver this year’s Baddiley lecture. It was an honour to meet Venki, who somehow managed to fit us in between Royal Society committee meetings and a chat with the Science minister! He impressed us all with a phenomenal talk discussing how he solved the structure of the mitochondrial ribosome using cryo electron microscopy. Wow – cryo EM has truly moved beyond blob-ology! One thing that really struck me about Venki is how humble he is; despite being so incredibly successful and lauded, there’s not a trace of ego on the guy; something for us all to aspire to.”

Venki illustrates how the ribosome works

Venki illustrates how the ribosome works

Seamus Holden, University Research Fellow

“It was incredibly cool to hear about Venki’s work first hand. The enormity of his achievement became clear when he showed a single slide with the dozens of conformations of the ribosome’s catalytic cycle and indicated that there were structures available for the majority of them! And what was humbling was that Venki did not seem at all interested in dwelling upon his past successes. Rather, he briskly moved past this slide onto his current work regarding mitochondrial ribosomes which was both cutting-edge but also somewhat raw because of its novelty. I found it inspiring to see a scientist of his stature still so driven to continue discovering and learning.”

Post lecture, Venki holds the gift he received from his hosts at ICaMB

Post lecture, Venki holds the gift he received from his hosts at ICaMB

Heath Murray, Royal Society URF

“For me Venki’s journey was an excellent advert for never giving up. Often as researchers (particularly at the start of our careers) we are encouraged to know when to call time on a set of experiments that are bogged down and not yielding answers. Pursuit of the next grant and the speed of some of our competitors unfortunately make it risky to continue to spend years and years believing in the same project that fails to show progress relatively quickly. Yet what an example Venki is, many years and many postdocs focusing on the same problem, persistence and belief in himself and his team has more than paid off. Inspirational.”

Suzanne Madgwick, Wellcome Trust Career Re-entry Fellow

ECRs at ICaMB: Solving 3D puzzles

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by Dr Paula Salgado

After nearly one year editing the ICaMBlog, the time has come for me to tell you about my science and work since I joined ICaMB almost 18 months ago.

The fact that it has been 18 months since I moved up North to establish my own research group seems to have snuck up on me… Don’t get me wrong, so much has happened that, if anything, it’s surprising it all took place in 1 and a half years. At the same time, the feeling of a new adventure is still there.

Science is a constant adventure to seek new knowledge, to understand new mechanism, to see new things. In my case, to see into the very core of life’s machines: proteins. I use X-ray protein crystallography to probe the structure of proteins. It’s a bit like solving a puzzle: fitting the pieces of information together until we have a 3D view of the protein.

It is actually fitting that my blog post is the first ICaMB publishes in 2014 as this is the International Year of Crystallography. I could write a lot about it, but for now, I’ll leave you with an amazing video made by the Royal Institution that explains it all – in cartoons! If you want to know more about Crystallography, the Ri has a great collection of videos there, including Prof Stephen Curry’s Friday Evening Discourse, which I strongly recommend.

Freezing protein crystals for data collection at Diamond Light Source. H&S warning: liquid nitrogen is a hazard and we do handle it safely. At this point, I was just dipping the crystals into a small volume, all other procedures handling larger volumes involve wearing appropriate protection.

As a protein crystallographer, I’ve always been interested in proteins that have a relevance to human disease and used this technique to understand their structure and function. In the last few years, I’ve worked on proteins from human pathogens associated with hospital acquired infections, particularly Clostridium difficile and Candida albicans. However, protein structures don’t necessarily give us all the answers and they must be complemented with biochemical studies, as well as in vivo experiments. So my long term goal has become to establish a Structural Microbiology group, where we focus on structure determination of key proteins and complexes involved in pathogenicity as well as on their functional in vivo characterisation.

This is a challenge as it means stepping out of my structural biology comfort zone into the world of microbiology and cell biology. Not that I haven’t stepped out of my comfort zone before – if anything, those are areas that featured strongly during my undergraduate training as a Biochemistry student at the University of Porto in Portugal. In those days, choosing to do protein crystallography as my undergraduate project was the big step into the unknown. A trend that continued as a post-doc, when I joined Dr Cota and Prof Mathews group, a Nuclear Magnetic Resonance (NMR) lab at Imperial College, learning a completely different approach to protein structure determination. And just before coming to ICaMB, I worked in Prof Fairweather’s microbiology lab and always tried to learn a bit about the techniques others were using. So the current idea of bringing structural biology and microbiology expertise together in my lab is the natural evolution of these experiences.

C. difficile cells (green rods) lining the microvilli of the human gut. © Wellcome Trust (CC-BY)

Since joining ICaMB, I’ve focused on 2 main projects, both involving proteins from C. difficile. This spore forming strict anaerobe is resistant to most antibiotics and colonises the gut of individuals whose microbiome has been disturbed by these drugs. It is the most prevalent cause of gastrointestinal infections in hospitals and is a major cause of morbidity and mortality in the hospital environment. Despite recent decreases in the number of deaths and infections as hygiene procedures have improved in the UK, over 1600 people died in England and Wales in 2012 due to C. difficile infections (CDI). It also causes a huge burden to health systems, with an estimated €3,000 million per annum costs in the EU.

C. difficile disease symptoms are caused by the toxins it releases in a process that has been extensively studied over the years. However, the mechanisms of colonisation of the gut and spore formation are poorly understood. So we have been focusing on proteins involved in these two mechanisms.

Firstly, I’ve been trying to determine the structure of SlpA, the main protein constituent in C. difficile S-layer. S-layer is a paracrystalline coat that covers the cell and is presumed to act like a defense mechanism, as well as being involved in gut colonisation. This work, initiated a few years ago in Prof Fairweather’s lab is now a joint collaboration between our two labs and Dr Fagan, at Sheffield University.

SlpA crystals viewed under polarised light (protein crystals are birefringent, unlike salt crystals)

 

 

As this protein has tendency to form 2D paracrystalline layers, getting well ordered 3D crystals required for X-ray crystallography has been a challenge, but I have now succeeded in obtaining good crystals. However, other hurdles still need to be overcome to get a structure – but we are getting there!

 

 

Our lab: Adam Crawshaw and Paula Salgado

Last year, Adam Crawshaw joined my group as a BBSRC Doctoral Training Programme (DTP) student and we started a new project, looking at a complex between two membrane proteins that are essential for spore formation. As spores are the infectious agents, revealing the molecular details sporulation is important to understand the pathogenicity and infection cycle of C. difficile.

SpoIIQ and SpoIIIAH localise at the membranes of the forming forespore. Green: Membrane; Red: SNAP-tagged proteins.

When spores are first formed, a small cell (forespore) is engulfed by the larger mother cell, physically isolating it from the environment and nutrients in the medium. So, for the forespore to fully mature, it needs a nurturing channel to the mother cell. The two proteins we are studying  – SpoIIQ from the forespore membrane and SpoIIIAH from the mother cell membrane – create this channel. We have already successfully produced recombinant versions of the proteins and shown their interaction in vitro. In collaboration with Prof Henriques at ITQB, Lisbon, we also established their localisation during C. difficile spore formation. Next: crystals! But we are also investigating potential enzymatic activity both in vitro and in vivo, to bring the structure and biology together.

It has been an exciting year and a half – a steep learning curve with many new tasks, from supervising students to managing a lab and teaching undergraduates and postgraduates. The adventure continues, with new challenges and exciting discoveries ahead.

 

Links

International Year of Crystallography http://www.iycr2014.org/

Royal Institution Crystallography gallery http://richannel.org/celebrating-crystallography

Office for National Statistics (Clostridium difficile data) http://www.ons.gov.uk/ons/rel/subnational-health2/deaths-involving-clostridium-difficile/2012/index.html

European Centre for Disease Intervention and Control on Clostridium difficile  http://www.ecdc.europa.eu/en/healthtopics/healthcare-associated_infections/clostridium_difficile_infection/pages/index.aspx

BBSRC Doctoral Training Programme http://www.ncl.ac.uk/fms/dtp/