I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXm-Versatile Macromolecular Crystallography microfocus
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Open Access
Abstract: Structure determination by X-ray diffraction is limited by crystal size and can be compromised by radiation damage when using very intense X-ray radiation. X-ray structure determination from partial diffraction data sets combined from multiple crystals is a potential solution, but its exploitation in chemistry and materials science is largely unrealized. Here we report the use of synchrotron radiation for multi-crystal X-ray diffraction (MCXRD) adapted for structure determination of metal-organic framework (MOF) materials with crystal dimensions too small for conventional single-crystal diffraction studies. We further show that radiation-induced chemical changes and degradation of diffraction quality can be alleviated. Our approach encompasses both rotation- and stationary-MCXRD measurements for 10 to 1000s of crystals with software-optimized combination of the multiple data sets. We report the crystal structures of six MOFs: MOF-919(Sc/Cu), MET-2, MIL-88B(Cr)-1,4-NDC, PCN-260(Sc), UiO-66, and UiO-66-MoO4 with unit cell dimensions ranging from 18−114 Å and crystal sizes from 0.5−480 µm3. This approach can address the challenges of structure determination in a regime of particle size and sample radiation sensitivity that lies between existing single-crystal X-ray diffraction and the emerging field of electron diffraction. MCXRD can provide accurate atomic-resolution structure determination for some of the most challenging cases in chemistry and materials science.
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Jan 2026
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VMXm-Versatile Macromolecular Crystallography microfocus
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Open Access
Abstract: Determining the structure of a protein is essential for understanding its function. However, X-ray crystallography becomes increasingly difficult as the diffracting power of crystals decreases with a decrease in crystal size. This challenge is further exacerbated by the fact that more complex targets tend to crystallize on smaller scales, and efforts to produce larger crystals often fail. Over recent years, serial crystallography techniques at synchrotrons and X-ray free electron lasers (XFEL) have been developed to enable structure determination from smaller crystals and to carry out time-resolved experiments.1 Unfortunately, these methods require large sample quantities, which can be difficult, costly, and time-consuming to produce, particularly for novel systems where little prior information is known. For crystals smaller than 300 nm, micro-electron diffraction (microED) has emerged as a solution for structure determination.2 However, it can be challenging to confirm that a sample is of the correct size for these experiments, and often, samples are too large, necessitating focused ion beam milling to achieve the required sample thickness.3, 4, 5
The Versatile Macromolecular Crystallography Microfocus (VMXm)6 beamline was specifically designed to address these challenges by enabling rotation data collection from samples smaller than 20 μm, requiring only minimal sample volumes. This has been achieved through novel mounting of crystals on cryo-electron microscopy grids, blotting away excess liquid,7 conducting data collections in vacuum, and matching the beamsize to the crystal size. These strategies limit the background scatter, allowing weak signal from the micro/nanocrystals to be detected. An additional advantage comes from collecting diffraction data at higher X-ray energies (∼21 keV) to exploit photoelectron escape, extending the lifetime of the crystals in the beam.8, 9 To date, successful X-ray diffraction measurements have been performed on protein crystals as small as ∼1.2 μm and chemical crystallography samples down to 800 nm.
In this work we will present the beamline, and the novel strategies adopted to obtain multicrystal data from micro- and nanocrystals. In particular we will focus on comparisons between data collected on more traditional synchrotron beamlines, as well as XFELs to highlight the impact these strategies have on the production of high quality diffraction data.
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Oct 2025
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VMXm-Versatile Macromolecular Crystallography microfocus
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Open Access
Abstract: X-ray diffraction (XRD) of microcrystals is signal-to-noise limited by the inherently weak diffraction. As such, Electron diffraction (ED) is increasingly used to measure diffraction data from submicron crystals, or those deemed too small for XRD due the stronger interaction of electrons with matter. However, many samples which are too thin for XRD are often too thick for ED using the currently available electron beam energies (<300 keV) and hence require thinning by focussed ion beam milling (FIB) which adds additional sample preparation steps. In addition to determining structures from nanocrystals, ED provides Coulomb potential data which are complementary to that obtained with XRD. As such ED data may be necessary to answer particular scientific questions.
The macromolecular crystallography beamline, VMXm, at Diamond Light Source, has been optimised for maximising the S:N in XRD experiments with a variable focus high-energy (>20 KeV) X-ray beam, with in-vacuum endstation and the use of low background cryoTEM grids for crystal mounting [1], [2]. This has allowed VMXm to collect high-resolution rotation data from single crystals measuring ∼1.2 μm which were only previously tractable using an X-ray Free Electron Laser [3]. This has pushed the amenable sample envelope at synchrotrons to new dimensions and perhaps near to the practical limit of XRD. Indeed, simulations have predicted the limit to be ∼0.5 μm thick in the case of lysozyme, assuming photoelectron escape [4]. This XRD beamline opens up the possibilities to directly compare XRD and ED datasets and understand the complementarity of these experiments.
In this work we present data from cubic human insulin crystals that have been thinned by FIB milling from ∼10 μm to various submicron thicknesses. 200 kV ED data were then collected from these lamellae before XRD data were measured from the same lamellae using VMXm. It was possible to obtain a complete XRD dataset to 2.45 Å using a 1.68 μm3 illuminated volume and a 2.04 Å ED dataset from the same 0.25 μm lamella. We have demonstrated that the data quality is comparable between ED and VMXm from the same crystal, while giving an opportunity to directly compare X-ray and electron derived maps. This includes the comparison of the radiation damage each experiment imparts on the sample [5] as well as the information content [6]. This work indicates that the usable sample envelope for synchrotron X-rays extends to much thinner samples than had been previously thought. It is also the first demonstration of ED and XRD measured from the same crystal volume enabling direct comparison of X-ray and electron derived data. Ultimately, the work will inform the design and use of high energy (MeV) ED instruments such as HeXI and how those can be complemented by XRD derived information from beamlines such as VMXm.
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Oct 2025
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VMXm-Versatile Macromolecular Crystallography microfocus
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Abstract: X-ray diffraction (XRD) of microcrystals is signal-to-noise limited by due to the inherently weak diffraction. Therefore, it is key that the beamline instrumentation and the sample itself introduce minimal noise. The VMXm beamline, at Diamond Light Source, has been optimised for maximising the S:N in experiments with a variable focus high-energy (>20 KeV) X-ray beam, with in-vacuum endstation and the use of low background cryoTEM grids for crystal mounting [1], [2]. This has allowed VMXm to collect high-resolution rotation data from single crystals measuring ~1.2 μm which were only previously tractable using an X-ray Free Electron Laser [3]. This has pushed the amenable sample envelope at synchrotrons to new dimensions and perhaps near to the practical limit of XRD. Indeed, simulations have predicted the limit to be ~0.5 μm thick in the case of lysozyme, assuming photoelectron escape [4].
Electron diffraction (ED) is frequently used to measure diffraction data from submicron crystals. Many samples which are too thin for XRD are often too thick for ED using the currently available electron beam energies (<300 keV) and hence require thinning by focussed ion beam milling (FIB). In addition to determining structures from nanocrystals, ED provides Coulomb potential data which are complementary to that obtained with XRD. As such ED data may be necessary to answer particular scientific questions.
In this work we present data from cubic human insulin crystals that have been thinned by FIB milling from ~10 μm to various submicron thicknesses. 200 kV ED data were then collected from these lamellae before XRD data were measured from the same lamellae using VMXm. It was possible to obtain a complete XRD dataset to 2.45 Å using a 1.68 μm3 illuminated volume and a 2.04 Å ED dataset from the same 0.25 μm lamella. We have demonstrated that the data quality is comparable between ED and VMXm from the same crystal, while giving an opportunity to directly compare X-ray and electron derived maps. This includes the comparison of the radiation damage each experiment imparts on the sample [5] as well as the information content [6]. This work indicates that the usable sample envelope for synchrotron X-rays extends to much thinner samples than had been previously thought. It is also the first demonstration of ED and XRD measured from the same crystal volume enabling direct comparison of X-ray and electron derived data. Ultimately, the work will inform the design and use of high energy (MeV) ED instruments such as HeXI and how those can be complemented by XRD derived information from beamlines such as VMXm.
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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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VMXm-Versatile Macromolecular Crystallography microfocus
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Anna J.
Warren
,
Jose
Trincao
,
Adam D.
Crawshaw
,
Emma V.
Beale
,
Graham
Duller
,
Andrew
Stallwood
,
Mark
Lunnon
,
Richard
Littlewood
,
Adam
Prescott
,
Andrew
Foster
,
Neil
Smith
,
Guenther
Rehm
,
Sandira
Gayadeen
,
Christopher
Bloomer
,
Lucia
Alianelli
,
David
Laundy
,
John
Sutter
,
Leo
Cahill
,
Gwyndaf
Evans
Open Access
Abstract: VMXm joins the suite of operational macromolecular crystallography beamlines at Diamond Light Source. It has been designed to optimize rotation data collections from protein crystals less than 10 µm and down to below 1 µm in size. The beamline has a fully focused beam of 0.3 × 2.3 µm (vertical × horizontal) with a tuneable energy range (6–28 keV) and high flux (1.6 × 1012 photons s−1 at 12.5 keV). The crystals are housed within a vacuum chamber to minimize background scatter from air. Crystals are plunge-cooled on cryo-electron microscopy grids, allowing much of the liquid surrounding the crystals to be removed. These factors improve the signal-to-noise during data collection and the lifetime of the microcrystals can be prolonged by exploiting photoelectron escape. A novel in vacuo sample environment has been designed which also houses a scanning electron microscope to aid with sample visualization. This combination of features at VMXm allows measurements at the physical limits of X-ray crystallography on biomacromolecules to be explored and exploited.
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Nov 2024
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I24-Microfocus Macromolecular Crystallography
VMXm-Versatile Macromolecular Crystallography microfocus
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Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Abstract: The world is full of treasure troves of hoarded scientific samples - everything from pressed plants in herbaria and pinned insects in museum drawers to rocks from the Moon and asteroids in air-tight vaults. Generations of scientists have contributed to collecting, preserving and studying such specimens, amassing an immense amount of knowledge about our natural world and how it changes over time. As we develop ever more powerful technologies to explore them, these historical samples can lead us to new discoveries, as an international team of researchers recently found. Their work, published in Nature Communications, began with a 70-year-old viral sample tracked down in a low temperature freezer. This viral sample was of a Nudivirus that are economically important in agriculture, where they are used as biological agents to control some insect pests. However, they can be pests themselves, damaging large-scale crustacean and insect farming efforts. Using X-ray crystallography, the research team solved the lattice structure of a polyhedrin (occlusion body) from Nudiviridae, which adds to our knowledge of how viruses use protein self-assembly to protect themselves from the environment.
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May 2024
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I24-Microfocus Macromolecular Crystallography
VMXm-Versatile Macromolecular Crystallography microfocus
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Jeremy R.
Keown
,
Adam D.
Crawshaw
,
Jose
Trincao
,
Loic
Carrique
,
Richard J.
Gildea
,
Sam
Horrell
,
Anna J.
Warren
,
Danny
Axford
,
Robin
Owen
,
Gwyndaf
Evans
,
Annie
Bézier
,
Peter
Metcalf
,
Jonathan M.
Grimes
Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Open Access
Abstract: Infectious protein crystals are an essential part of the viral lifecycle for double-stranded DNA Baculoviridae and double-stranded RNA cypoviruses. These viral protein crystals, termed occlusion bodies or polyhedra, are dense protein assemblies that form a crystalline array, encasing newly formed virions. Here, using X-ray crystallography we determine the structure of a polyhedrin from Nudiviridae. This double-stranded DNA virus family is a sister-group to the baculoviruses, whose members were thought to lack occlusion bodies. The 70-year-old sample contains a well-ordered lattice formed by a predominantly α-helical building block that assembles into a dense, highly interconnected protein crystal. The lattice is maintained by extensive hydrophobic and electrostatic interactions, disulfide bonds, and domain switching. The resulting lattice is resistant to most environmental stresses. Comparison of this structure to baculovirus or cypovirus polyhedra shows a distinct protein structure, crystal space group, and unit cell dimensions, however, all polyhedra utilise common principles of occlusion body assembly.
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Jul 2023
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VMXm-Versatile Macromolecular Crystallography microfocus
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Leila T.
Alexander
,
Janani
Durairaj
,
Andriy
Kryshtafovych
,
Luciano A.
Abriata
,
Yusupha
Bayo
,
Gira
Bhabha
,
Cécile
Breyton
,
Simon G.
Caulton
,
James
Chen
,
Séraphine
Degroux
,
Damian C.
Ekiert
,
Benedikte S.
Erlandsen
,
Peter L.
Freddolino
,
Dominic
Gilzer
,
Chris
Greening
,
Jonathan M.
Grimes
,
Rhys
Grinter
,
Manickam
Gurusaran
,
Marcus D.
Hartmann
,
Charlie J.
Hitchman
,
Jeremy R.
Keown
,
Ashleigh
Kropp
,
Petri
Kursula
,
Andrew L.
Lovering
,
Bruno
Lemaitre
,
Andrea
Lia
,
Shiheng
Liu
,
Maria
Logotheti
,
Shuze
Lu
,
Sigurbjorn
Markusson
,
Mitchell D.
Miller
,
George
Minasov
,
Hartmut H.
Niemann
,
Felipe
Opazo
,
George N.
Phillips
,
Owen R.
Davies
,
Samuel
Rommelaere
,
Monica
Rosas‐lemus
,
Pietro
Roversi
,
Karla
Satchell
,
Nathan
Smith
,
Mark A.
Wilson
,
Kuan‐lin
Wu
,
Xian
Xia
,
Han
Xiao
,
Wenhua
Zhang
,
Z. Hong
Zhou
,
Krzysztof
Fidelis
,
Maya
Topf
,
John
Moult
,
Torsten
Schwede
Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Open Access
Abstract: We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.
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Jul 2023
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VMXm-Versatile Macromolecular Crystallography microfocus
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Lennart
Brewitz
,
Leo
Dumjahn
,
Yilin
Zhao
,
C. David
Owen
,
Stephen M.
Laidlaw
,
Tika R.
Malla
,
Dung
Nguyen
,
Petra
Lukacik
,
Eidarus
Salah
,
Adam D.
Crawshaw
,
Anna J.
Warren
,
Jose
Trincao
,
Claire
Strain-Damerell
,
Miles W.
Carroll
,
Martin A.
Walsh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.
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Feb 2023
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