Structural & Molecular Biology

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Principal Investigator: Emma Springate

Artemis investigates ultrafast electron dynamics in condensed matter and gas-phase molecules, and for coherent lensless imaging.

Artemis is based on high repetition rate, few optical cycles and widely tuneable laser sources, and ultrafast XUV (10-100 eV) pulses produced through high harmonic generation. We exploit the femtosecond time-resolution afforded by harmonics to use them as ultrafast probes of electron dynamics. Our key technique is time-resolved photoelectron spectroscopy with XUV high harmonic probe pulses and we have three dedicated end-stations for gas- and solid-phase experiments. We also exploit the spatial coherence of the XUV to use coherent diffractive imaging techniques.

Artemis will move across the campus to RCaH in late 2018 as part of a major upgrade funded by STFC and BEIS. The upgrade adds a new laser system (a joint purchase with Ultra) and a new XUV beamline. The new laser will use OPCPA technology to provide mid-infrared pulses at 100 kHz repetition rate.

For Artemis, the mid-infrared will enable the generation of higher photon energy XUV pulses and the higher repetition rate allows smaller samples to be studied. For Ultra, the appeal is the ability to provide broader spectral coverage at high repetition rates, for faster data acquisition, and more efficient generation of mid- to far-IR pulses.

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Principal Investigator: Konstantinos Beis

Our group is interested in the structural and functional characterisation of membrane proteins that are involved in bacterial multidrug resistance and human diseases. We use protein X-ray crystallography for their structure determination. We are also involved in developing methods for membrane protein crystal data collection including phasing.

These are some of our research areas.

Multidrug resistance
The group’s primary interest is to understand how bacteria use ABC transporters and multidrug efflux pumps to export structurally unrelated antibiotics out of the cell, thus conferring them with resistance. From their crystal structures, we want to identify novel molecules that can inhibit their function.

Antibacterial peptides
We are interested in exploiting novel antibacterial peptides that bacteria produce under nutrient starvation for survival that could potentially be used as treatments for multidrug resistance. To date, we have determined the structure and function of such peptides in complex with outer membrane receptors.

Human disease
Human receptors and transporters are involved in several processes and mutations can lead to serious diseases. Transporters are also involved in the export of anticancer drugs during chemotherapy. We are investigating their structure and function to understand their role in human disease.

Principal Investigator: Dr David Scott

The Biophysical Methods Group is led by Dr David Scott (University of Nottingham), Senior Molecular Biology and Neutron Fellow (ISIS/STFC).

We develop and use a variety of biophysical methods to characterise the interactions of biological macromolecules in solution and in the cell. These include proteins (soluble and membrane-bound), protein/DNA complexes and nanoparticles. We have expertise in analytical ultracentrifugation, calorimetry, structural methods and various spectroscopies.

Projects include:

• Using neutrons to investigate biological macromolecules
• Bacterial silver resistance
• Ube3A and its interactions
• CryoCLEM
• CCP-SAS
• Development of analytical ultracentrifuge and small-angle scattering theory

Collaborative work

We are always interested in new collaborations with groups and individuals, both nationally and internationally.

Collaborations with doctoral training programmes

We are an active participant of the University of Nottingham BBSRC DTP and the MRC IMPACT DTP (Nottingham/Birmingham/Leicester), with students from both schemes placed in our group. Through RCaH, we can act as supervisors on any other collaborative DTP/CDT.

Fellowship placement

RCaH is a very well resourced, friendly and collaborative environment to place a fellowship. Within the group we already have visiting scientists  with their own fellowships, but are always happy to discuss placements or sponsorships of fellowships. Please contact Dr David Scott (dave.scott@rc-harwell.ac.uk) in the first instance to discuss any projects.

Principal Investigator: Dr Stephen Carr

Biological catalysis underpins all cellular processes and is reliant on the activity of many different enzymes, whose functions allow complex chemistry to be performed in water at ambient temperatures. We use a combination of X-ray crystallography, electron microscopy, biophysical methods and biochemistry to investigate how a variety of different enzymes work. Areas of interest include the function of metalloproteins (particularly hydrogenases), protein-DNA interactions and the role of ubiquitin ligases during development .

Hydrogenases

[NiFe]-Hydrogenases are complex metalloenzymes containing nickel and iron atoms at their active site, which allow bacteria to use hydrogen as an energy source. This is achieved by enzymatic splitting of hydrogen gas into protons and electrons. We use structural biology, electrochemistry and IR spectroscopy (in collaboration with Professors Fraser Armstrong and Kylie Vincent, University of Oxford) to understand how the enzyme influences the properties of nickel and iron ions bound within the protein in order to split or produce hydrogen. Analysis of variant proteins containing mutated amino acids at the active site of the protein has allowed us to propose a novel mechanism for how [NiFe]-hydrogenases can achieve this.

We are pioneering the technique of single-crystal IR spectroscopy coupled with electrochemical control of the redox state of the crystallised protein to trap and determine the structures of intermediates of the catalytic cycle. This will allow a complete understanding of how these enzymes function in the cell. The active site may also act as inspiration for the design of novel, inexpensive chemical catalysts based on Ni/Fe that will be essential for the success of the future hydrogen economy.

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Principal Investigator: Eugene Krissinel

CCP4 exists to produce and support a world-leading, integrated suite of programs that allows researchers to determine macromolecular structures by X-ray crystallography and other biophysical techniques. CCP4 aims to develop and support the development of cutting-edge approaches to experimental determination and analysis of protein structure, and integrate these into the suite.

CCP4 is a community-based resource that supports the widest possible researcher community, embracing academic, not-for-profit and for-profit research. CCP4 aims to play a key role in the education and training of scientists in experimental structural biology. It encourages the wide dissemination of new ideas, techniques and practice.

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Principal Investigator: Dr David Clarke

The CLF is the UK’s national laser facility and offers access to advanced laser technologies ranging from extremely high power lasers for investigating matter under extreme conditions, to spectroscopy and imaging facilities for life sciences, chemistry and materials research. RCaH houses two of the CLF’s facilities, ULTRA and Octopus. Both facilities are operated by STFC and have received considerable investment from BBSRC and MRC. Access to the facilities for UK academics is free at the point of use, via a peer-reviewed proposal mechanism. Various routes are available for access by industrial users.

ULTRA combines laser, detector and sample manipulation technology to study molecular dynamics to address scientific problems in the physical and life sciences. A range of ultrafast light sources provides unprecedented flexibility to combine multiple beams, multiple colours (UV to mid-IR), mixed timing patterns (fs-µs) and pulse length. ULTRA is one of the world’s most sensitive time-resolved spectrometers and is used to investigate dynamics of complex biological systems such as proteins. ​

Octopus is an advanced imaging facility containing a mixture of in-house-built and commercial systems, offering a range of imaging techniques. These include several modes of multidimensional single molecule microscopy, and structure determination in fixed cells, at ~5nm resolution via fluorescence localisation with photobleaching (FLimP)). Other techniques include super-resolution microscopy (STORM, PALM, SIM, STED), confocal microscopy (FLIM, FRET, and multiphoton), and light sheet microscopy.

ULTRA and Octopus will soon be joined in RCaH by Artemis, the CLF’s facility for ultrafast XUV science. Experiments on Artemis use high harmonics to investigate ultra-fast electron dynamics in condensed matter and gas-phase molecules, and for coherent lensless imaging.

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The Collaborative Computational Project for electron cryo-microscopy (CCP-EM) supports users and developers in biological cryoEM, including single-particle analysis, tomography and diffraction.

The three principal aims are to:

• Build a UK community for computational aspects of cryo-EM. Provide a focus for the cryo-EM community to discuss computational issues, and to interact with the broader structural biology community.
• Support the users of software for cryo-EM through dissemination of information on available software and directed training. Collate, develop and distribute a CCP-EM software suite to help users processing cryoEM data.
• Provide support for software developers including porting, testing and distribution of software.

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Principal Investigator: Peijun Zhang

This research programme is aimed at obtaining an integrated, atomistic understanding of the molecular mechanisms of large viral and cellular protein complexes and assemblies by developing novel technologies for high-resolution cryo-EM and cryo-ET, with advanced, complementary methods for biological analysis and computational modelling. Such large systems have challenged the limits of structural biology methods.

Cryo-EM/cryo-ET technology development

Driven by biological enquiries, we are developing cutting-edge methods and technologies, such as those inspired by bottlenecks that we have had to overcome, towards:

1. atomic or near-atomic resolution in molecular cryo-ET by sub-tomogram averaging and classification
2. in situ structure determination using cryo-FIB technologies and cellular cryo-ET
3. correlative and integrative structural biology using light and electron microscopy.

We will apply these technologies to move towards a clearer understanding of the intricate interplay between pathogens and host cells. These efforts will have a broad impact well beyond this research.

Human pathogens, such as HIV-1 and pathogenic bacterial cells

We are interested in HIV-1 capsid assembly, maturation and its interactions with host cellular factors that inhibit or enhance viral infectivity. We are also working to understand the molecular mechanism underpinning the remarkable bacterial chemotaxis sensory systems that are crucial for colonisation and infection. Understanding the structural and dynamics details of these large systems, at atomic level, is critical for developing new anti-HIV/AIDS and antimicrobial drugs.

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Principal Investigator: Ralf Flaig

Epigenetic modifications (reversible, heritable genetic changes that occur without changes in DNA sequence) lead to chromatin remodelling, altered gene expression and changes in the cellular phenotype. They have therefore been linked to a number of pathologies, including cancer, cardiac disease, neurological disorders, infections and inflammatory diseases.

One epigenetic process is histone modification. This is important because gene regulation in eukaryotes requires the coordinate interaction of chromatin-modulating proteins with specific transcription factors. Gene activation and repression are specifically regulated by histone modification at distinct residues. Understanding these mechanisms can provide new and specific therapies for pathologies caused by these processes.

Structural characterisation and drug design of epigenetic regulators

Our research projects focus on two protein families that have been identified as potential therapeutic targets. One aim is to investigate the structure and function of proteins binding to histone-modifying proteins. A second aim is to structurally characterise potential inhibitors of lysine demethylases and histone acetylases and elucidate the structural and functional relationships between these proteins and potential small molecule inhibitors. This will help the process of using structure-based optimisation to find potential drug molecules for pre-clinical assessment. The project involves the use of established biochemistry and molecular biology techniques and structure determination by X-ray crystallography.

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Principal Investigator: Professor Philip Davies

We offer access to state-of-the-art spectrometers for photoelectron spectroscopy in our main laboratory based at RCaH. Together with our partner hubs offering specialist analysis at Cardiff University, UCL and the University of Manchester, the Harwell XPS service provides access to a wider range of XPS analysis methods than previously available to UK academia and industry, including:

• XPS and UPS
• angle resolved XPS (ARXPS)
• XPS imaging
• ion scattering spectroscopy (ISS/LEIS)
• cluster and monotomic ion depth profiling
• high energy XPS and near ambient pressure (NAP) XPS
• high temperature and pressure treatments.

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Principal Investigator: Armin Wagner

We have designed, constructed and commissioned the novel long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source.

This unique instrument allows X-ray diffraction experiments in vacuum in a wavelength range of 1.2 – 5.9 A with a semi-cylindrical X-ray diffraction detector.

We are pioneering experiments using this wavelength range for experimental phasing experiments exploiting weak anomalous signals from native proteins without labelling and using anomalous contrast to identify light atoms of biological significance such as P, S, K and Ca.

Our group has a strong background in structural biology, sample handling and instrumentation for synchrotron and XFEL experiments.

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Principal Investigator: Professor Pavel Matousek

Research focuses on the development of advanced Raman spectroscopy ‘through-barrier’ methods for non-invasive disease diagnosis (e.g. breast cancer and bone disease diagnosis), security screening (airport security, detection of hazardous materials), cultural heritage (intact analysis of painted layers) and pharmaceutical analysis (quality control) and the development of advanced nano-photonic concepts for personalised, single-session cancer diagnosis and treatment.

A number of approaches used are based around spatially offset Raman spectroscopy (SORS), a patented concept originating from research on the CLF-RCaH ULTRA facility. These activities are carried out with numerous external partners including Exeter University (Professor Nicholas Stone), UCL (Professor Allen Goodship), STFC (Professor Tony Parker) and CNR-ICVBC in Milan (Dr Claudia Conti).

The research also has a strong commercial component: for example, with the co-founding in 2008 of Cobalt Light Systems Ltd. Cobalt developed commercial SORS scanners that are deployed at more than 75 airports to scan liquids in bottles (e.g. baby milk, medicine) and 30 pharmaceutical companies worldwide in quality control, inspecting finished products and incoming raw materials by intact means. In 2017, Cobalt was acquired by Agilent for £40 million, forming in Oxford its global centre for Raman spectroscopy.

Pavel is an Honorary Professor at University College London and an STFC Senior Fellow (main role) and his research is conducted within LSF-CLF unit at RCaH. His main research funders include STFC and EPSRC.

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Principal Investigator: Dr Andrew Quigley

The Membrane Protein Laboratory (MPL) is a Wellcome Trust funded user facility that is part of the macromolecular crystallography village at Diamond Light Source. The MPL is focused on the efficient determination of relevant membrane protein structures by combining recently developed high-throughput technologies for protein production and crystallisation with the X-ray diffraction data collection system at Diamond. Since 2010, the MPL has supported well over 100 applications from all around the world to use its facilities and has contributed to the solving of one membrane protein structure a year since 2012.

The MPL, located at RCaH, has access to state-of-the-art equipment necessary for membrane protein expression, purification and sample preparation for X-ray crystallography and cryo-electron microscopy. All MPL equipment and methodologies are available to RCaH scientists, visitors and collaborators and we have dedicated research scientists available to support all users of our facility.

Applications for access to the MPL should be made by following the instructions on our website.

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Principal Investigator: Allen Orville

Life is dynamic and function is not compatible with the cryogenic conditions used in traditional crystallography and cryo-EM. We focus on X-ray free-electron lasers (XFELs), which are located in the USA, Japan, Korea, Germany and Switzerland.

Even submicron size crystals yield high-quality structures because of the unparalleled intensity of these XFEL facilities. Serial femtosecond crystallography (SFX) methods exploit slurries of microcrystals, the XFEL fs pulse duration, and are conducted at ambient temperature. This allows us to couple structural biology with functional dynamics. We are also developing strategies for dynamic structural biology at Diamond’s VMXi beamline.

New X-ray sources create new opportunities; as a result, we are entering an era of dynamic structural biology. This is as much a concept, as a set of tools to collect as much data as possible, from every sample and X-ray pulse, and enables one to create atomic resolution ‘movies’ of macromolecules engaged in catalysis. Thus, time-resolved crystallography, a long-standing frontier challenge for the field, is achievable with serial methods at XFELs and at advanced synchrotron beamlines.

Principal Investigator: Professor Ray Owens

Protein Production UK (PPUK) is a initiative that began in the Research Complex that addresses sample preparation for challenging proteins and multicomponent complexes. PPUK aims to develop new and innovative methods and to work in partnership with academic groups and industry to enable uptake of the technology.

PPUK is now part of the Rosalind Franklin Institute and closely linked to the Central Facilities on the Harwell campus but maintains its relationship with Research Complex. It aims to develop new and innovative methods and to work in partnership with academic groups and industry to enable uptake of the technology.

PPUK has an established pipeline for protein production based on the technology developed by the Oxford Protein Production Facility, involving construction of multi-purpose vectors (e.g. for producing complexes, and membrane proteins), high-throughput expression screening in different cell hosts (microbial, insect and mammalian cells) combined with scale-up to multi-litre culture volumes and downstream processing.

New synergistic technology platforms will be developed to underpin the RFI and provide new capabilities to the user community, including production of single-domain antibodies and chaperones for the structural and functional analysis of proteins. In particular, this includes the stabilisation of flexible channels or complexes and the preparation of complex samples for and presentation to the beam in cryo-EM.

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Principal Investigator: Martin Walsh

Our research uses a targeted structural and functional approach to understanding at the molecular level how bacteria cause disease.

We have focused our efforts on bacterial pathogens that continue to pose a significant health risk to the very young and elderly. Within these pathogens, we have identified a number of vital processes to characterise and assess as targets for drug discovery to combat the rise in antimicrobial resistance (AMR).

Our interests lie predominantly in understanding the mechanisms used by bacterial pathogens to adhere to host cell surfaces and the structure, makeup and regulation of bacterial biofilms.

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Principal Investigator: Frank von Delft

The XChem Lab in the RCaH is an extension of the XChem initiative, a joint lab spanning Diamond and SGC-Oxford, which developed and runs Diamond’s very successful XChem facility for crystal-based fragment screening. At the RCaH, we are developing methodology in computational and synthetic chemistry to progress XChem hits to credible potency rapidly and cheaply.

The XChem facility allows hundreds of fragment molecules to be screened against crystallised protein targets, much faster than previously but with the high sensitivity of the crystallographic experiment. The readout thus yields directly the high-resolution structural information, i.e. specific configurations and positions of the bound fragments, needed to compute follow-up molecules.

To help analyse this uniquely rich dataset, we are building cloud- and HPC-based computational chemistry tools that can both enumerate synthetically and/or logistically accessible follow-up compounds and aid the decision process as to which subset to obtain, from both suppliers and chemist collaborators. In the first instance, we seek to capture and guide best practice in what currently is tedious and requires arcane expertise. This work forms the foundation of the nascent CCP-CompMedChem initiative.

To investigate how to expand the number of compounds that can be accessed practically, we are exploring how robotic synthesis can be combined with the XChem experiment to allow evaluation of hundreds rather than tens of follow-up compounds. The workhorse is currently the laughably cheap ($4,000) Opentrons liquid-handling robot.

This work aligns exactly with the chemistry theme of the Rosalind Franklin Institute.