I24-Microfocus Macromolecular Crystallography
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Abstract: Observing transient structural intermediates remains a central challenge in enzymology. Little is known about these despite their pivotal role in catalytic function. Time-resolved serial crystallography at synchrotron and X-ray free electron laser (XFEL) sources offers a promising avenue to capture these dynamic events. However, experimental success hinges on rigorous control of crystallisation, ligand delivery, and integration with beamline infrastructure. This thesis explores the use of droplet microfluidic and microcrystal strategies to address these bottlenecks and establish pipelines for future time-resolved studies.
A high-throughput crystallisation system was developed to generate uniform microcrystals within discrete aqueous droplets, leveraging a seeding strategy to overcome the low probability of nucleation at diminishing volumes. In parallel, a droplet micromixing device was engineered to initiate ligand-triggered reactions on millisecond timescales by exploiting convection within droplets as a means for rapidly mixing microcrystals with ligands. Flow parameters and mixing efficiency were characterised, followed by iterative design and fabrication of a X-ray transmissible device suitable for deployment at a synchrotron beamline.
Arabidopsis thaliana Pyridoxal 5'-phosphate synthase subunit 1.3 (AtPdx1.3) microcrystal slurries were validated for time-resolved studies using static serial femtosecond crystallography (SFX) at the SPring-8 angstrom compact free electron laser (SACLA). High-resolution radiation damage-free structures of apo and ligand-bound AtPdx1.3 were obtained at room temperature, representing the first XFEL structures of this enzyme. Diffraction from 20 μm crystals yielded resolution comparable to or better than previous larger crystals at cryogenic temperatures. Notably, only minimal structural differences were observed relative to cryotrapped structures, indicating strong conformational consistency. Soaking protocols enabled rapid ligand incorporation, capturing R5P, PLP and the crucial I320 intermediate within 15 minutes. These result establish robust workflows for intermediate state trapping and future dynamic studies. Taken together, the platforms developed in this thesis represent a significant step towards realising dynamic structural studies of enzymes at synchrotron and XFEL sources.
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Nov 2025
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I24-Microfocus Macromolecular Crystallography
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Peter
Smyth
,
Sofia
Jaho
,
Lewis J.
Williams
,
Gabriel
Karras
,
Ann
Fitzpatrick
,
Amy J.
Thompson
,
Sinan
Battah
,
Danny
Axford
,
Sam
Horrell
,
Marina
Lucic
,
Kotone
Ishihara
,
Machika
Kataoka
,
Hiroaki
Matsuura
,
Kanji
Shimba
,
Kensuke
Tono
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Shigeki
Owada
,
Michael A.
Hough
,
Jonathan A. R.
Worrall
,
Robin L.
Owen
Diamond Proposal Number(s):
[18565, 28583, 27313]
Open Access
Abstract: Time-resolved X-ray crystallography is undergoing a renaissance due to the development of serial crystallography at synchrotron and XFEL beamlines. Crucial to such experiments are efficient and effective methods for uniformly initiating time-dependent processes within microcrystals, such as ligand binding, enzymatic reactions or signalling. A widely applicable approach is the use of photocaged substrates, where the photocage is soaked into the crystal in advance and then activated using a laser pulse to provide uniform initiation of the reaction throughout the crystal. This work characterizes photocage release of nitric oxide and binding of this ligand to two heme protein systems, cytochrome c′-β and dye-decolourizing peroxidase B using a fixed target sample delivery system. Laser parameters for photoactivation are systematically explored, and time-resolved structures over timescales ranging from 100 µs to 1.4 s using synchrotron and XFEL beamlines are described. The effective use of this photocage for time-resolved crystallography is demonstrated and appropriate illumination conditions for such experiments are determined.
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Sep 2025
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I24-Microfocus Macromolecular Crystallography
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Abstract: Time-resolved diffraction is becoming an established technique in large-scale facilities such as synchrotrons and XFELs. Alternative sources such as plasma X-ray source (PXS, Fig. 1A) [1] offer attractive pulse durations of units of picoseconds with lower operational cost than XFEL facilities. However, the downsides of PXS sources are the unstable flux and comparatively low brilliance. The latter may be overcome by a stroboscopic [2] or multiplexing [3] approaches, however, the majority of macromolecular samples are radiation sensitive and undergo irreversible reactions limiting the application of stroboscopic data collection.
Hadamard Time-Resolved Crystallography (HATRX) is a multiplexing technique, where diffraction is measured as an ensemble of individual time points (Fig. 1B). The individual time-resolved data are then reconstructed using the Hadamard transform. Multiplexing requires the ability to measure diffraction at distinct time-points utilising either detector-gating or a pulsed source. We present initial results demonstrating radiation damage during a HATRX experiment using the detector-gating technique at beamline I24 (Diamond Light Source). The necessary pulse-sequences may be introduced onto the driving laser of the PXS, which is advantageous over detector gating since the sample is only exposed to X-rays when data are being collected. We discuss the potentials and pitfalls of the PXS for HATRX studies.
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Jun 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXm-Versatile Macromolecular Crystallography microfocus
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Diamond Proposal Number(s):
[26803, 34438, 35338]
Abstract: Due to radiation damage, the majority of metalloproteins structures are incorrect. Radiation damage in X-ray crystallography manifests itself either globally or at specific radiation sensitive sites. Global damage can be monitored from data processing statistics, whereas specific damage is more clandestine and presents as structural changes within the electron density. Serial crystallography using an X-ray free-electron laser promises a pseudo zero dose structure, however, the paucity of beamlines means beamtime is highly competitive. Method development, therefore, is required to collect low-dose structures using synchrotron X-ray crystallography. One dose-reducing phenomenon is photoelectron escape, where the generated photoelectrons escape the crystal volume before depositing their energy.
This thesis conducted the first serial crystallography experiment at VMXm (Diamond Light Source, UK), where photoelectron escape is significant for the targeted microcrystal sizes. An oxidised iron intermediate in myoglobin, Compound II, was tested as FeIV-oxo “ferryl” intermediates, which are known to be particularly susceptible to radiation damage. Despite not being a formal heme peroxidase, myoglobin is an excellent model for testing dose-limiting techniques. An NADP+-specific glyceraldehyde 3’-phosphate dehydrogenase from the enteric pathogen Helicobacter pylori was also investigated. NADP+-specificity is unusual amongst GAPDHs and are therefore poised for therapeutic targets. The kinetics of GAPDHA were investigated, and amongst the first structures of an NADP+-specific GAPDH outside of photosynthetic organisms are reported. An underreported form of radiation damage was observed. Therefore, a transition to microcrystals for a prospective dose-series and time-resolved investigation was performed.
A Mix and Quench Microcrystal Reactor was developed to initiate a reaction within microcrystals with rapid mixing and to trap intermediates by quenching in liquid ethane. Current systems exist for time-resolved crystallography or time-resolved cryoEM; however, a system was developed to react and spray microcrystals onto a TEM grid for use on the specific goniometry at VMXm.
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May 2025
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[33014, 35357]
Open Access
Abstract: The increasing availability of ultrabright Light Sources is facilitating the study of smaller crystals at faster timescales but with an increased risk of severe X-ray damage, leading to developments in multi-crystal methods such as serial crystallography (SX). SX studies on crystals with small unit cells are challenging as very few reflections are recorded in a single data image, making it difficult to determine the orientation matrix for each crystal and thus preventing the combination of the data from all crystals for structure solution. We herein present a Small-Rotative Fixed-Target Serial Synchrotron Crystallography (SR-FT-SSX) methodology, in which rotation of the serial target through a small diffraction angle at each crystal delivers high-quality data, facilitating ab initio unit cell determination and atomic-scale structure solution. The method is benchmarked using microcrystals of the small-molecule photoswitch sodium nitroprusside dihydrate, obtaining complete data to dmin = 0.6 Å by combining just 66 partial datasets selected against rigorous quality criteria.
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Nov 2024
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[260673]
Open Access
Abstract: Structure-based drug design is highly dependent on the availability of structures of the protein of interest in complex with lead compounds. Ideally, this information can be used to guide the chemical optimization of a compound into a pharmaceutical drug candidate. A limitation of the main structural method used today – conventional X-ray crystallography – is that it only provides structural information about the protein complex in its frozen state. Serial crystallography is a relatively new approach that offers the possibility to study protein structures at room temperature (RT). Here, we explore the use of serial crystallography to determine the structures of the pharmaceutical target, soluble epoxide hydrolase. We introduce a new method to screen for optimal microcrystallization conditions suitable for use in serial crystallography and present a number of RT ligand-bound structures of our target protein. From a comparison between the RT structural data and previously published cryo-temperature structures, we describe an example of a temperature-dependent difference in the ligand-binding mode and observe that flexible loops are better resolved at RT. Finally, we discuss the current limitations and potential future advances of serial crystallography for use within pharmaceutical drug discovery.
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Sep 2024
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I24-Microfocus Macromolecular Crystallography
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Abstract: X-ray crystallography is an important biological research technique that has provided most of the structures in the Protein Data Bank. An inherent challenge of crystallography is that the X-rays which produce diffraction images also cause damage to the crystal, resulting in loss of diffraction quality as the absorbed dose increases. To minimise this effect, serial crystallography techniques have been developed to spread dose over many crystals, by collecting a small amount of data, usually only one diffraction image, from each. This is especially beneficial for metalloproteins since metal centres are particularly susceptible to site-specific radiation damage and are important to the function of many proteins. The benefits of serial crystallography include the ability to perform experiments at room temperature, where proteins are more likely to be in physiologically relevant conformations, rather than at the cryogenic temperatures usually used to limit radiation damage. Room temperature also allows increased diffusion and molecular motions, so reactions can take place within protein crystals. These can be investigated with time-resolved serial experiments, capturing structures of short-lived intermediate states of a protein undergoing a dynamic process. An important part of any time-resolved experiment is reaction initiation, including ensuring the reaction begins in the whole sample at the same time.
This thesis describes the development of a method for time-resolved serial crystallography using the fixed-target chip system available on beamline I24 at Diamond Light Source, which is also applicable to serial femtosecond crystallography at X-ray free electron laser (XFEL) sources. These experiments explore the structures of highly radiation-sensitive metalloproteins, including time-resolved studies of nitric oxide binding in the haem site using a UV laser pump-probe technique and a photocaged substrate molecule.
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Jun 2024
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[27314]
Open Access
Abstract: Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.
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Apr 2024
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I24-Microfocus Macromolecular Crystallography
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Rachel
Bolton
,
Moritz M.
Machelett
,
Jack
Stubbs
,
Danny
Axford
,
Nicolas
Caramello
,
Lucrezia
Catapano
,
Martin
Maly
,
Matthew J.
Rodrigues
,
Charlotte
Cordery
,
Graham J.
Tizzard
,
Fraser
Macmillan
,
Sylvain
Engilberge
,
David
Von Stetten
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Jonathan A. R.
Worrall
,
Jeremy S.
Webb
,
Mike
Zubkov
,
Simon
Coles
,
Eric
Mathieu
,
Roberto A.
Steiner
,
Garib
Murshudov
,
Tobias E.
Schrader
,
Allen M.
Orville
,
Antoine
Royant
,
Gwyndaf
Evans
,
Michael A.
Hough
,
Robin L.
Owen
,
Ivo
Tews
Diamond Proposal Number(s):
[15722, 14493, 23570]
Open Access
Abstract: The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump–probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.
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Mar 2024
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I24-Microfocus Macromolecular Crystallography
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James
Birch
,
Tristan O. C.
Kwan
,
Peter J.
Judge
,
Danny
Axford
,
Pierre
Aller
,
Agata
Butryn
,
Rosana
Reis
,
Juan F.
Bada Juarez
,
Javier
Vinals
,
Robin L.
Owen
,
Eriko
Nango
,
Rie
Tanaka
,
Kensuke
Tono
,
Yasumasa
Joti
,
Tomoyuki
Tanaka
,
Shigeki
Owada
,
Michihiro
Sugahara
,
So
Iwata
,
Allen M.
Orville
,
Anthony
Watts
,
Isabel
Moraes
Diamond Proposal Number(s):
[19152]
Open Access
Abstract: Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.
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Oct 2023
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