|
|
Hayden
Fisher
,
Emma J.
Sutton
,
Robert J.
Oldham
,
Richard T.
Bradshaw
,
Patrick J.
Duriez
,
Björn
Frendéus
,
Gunilla
Larsson
,
Giusi
Manfredi
,
Marisa L.
Martin-Fernandez
,
Ian
Mockridge
,
Sarah R.
Needham
,
Daniel J.
Rolfe
,
Christian
Orr
,
Kallum
Patel
,
Ali
Roghanian
,
Alex
Simpson
,
Mark D.
Tully
,
Ingrid
Teige
,
Ulla-Carin
Tornberg
,
Christopher J.
Tynan
,
Abigail
Pendower
,
Jinny
Kim
,
Ariel
Tennenhouse
,
Sarel J.
Fleishman
,
Jonathan W.
Essex
,
Ivo
Tews
,
Mark S.
Cragg
Open Access
Abstract: The inhibitory human Fc gamma receptor, hFcγRIIB, is a key mediator of humoral immunity and regulator of antibody-mediated effector function. hFcγRIIB function can be modulated by anti-hFcγRIIB antibodies. We demonstrate that agonistic, but not antagonistic, antibodies reduce hFcγRIIB mobility in the plasma membrane, associated with receptor clustering and redistribution into lipid rafts. Agonists display lower affinity binding with higher off rates compared with antagonists. Using crystallographic structure determination and alanine-scanning mutagenesis, we show that epitopes targeted by agonistic and antagonistic antibodies are overlapping but distinct. Using small-angle X-ray scattering (SAXS) and molecular dynamics simulations, we demonstrate that agonists nucleate more compact receptor complexes. Through their high off rates, we propose that agonists facilitate a catch-and-release mechanism that promotes receptor clustering and subsequent activation. By contrast, antagonists adopt a binding geometry that prevents effective clustering, with their low off rate reducing receptor disengagement and subsequent clustering. These findings provide key principles underpinning agonism versus antagonism of immunomodulatory receptors.
|
Jul 2026
|
|
|
|
Open Access
Abstract: Online UV–Vis absorption spectroscopy is often used during crystallographic data collection to characterize chromophores within proteins. This includes the study of electron-rich or redox-active enzyme intermediates and cofactors that are prone to specific radiation damage, necessitating suitable low-dose data-collection strategies. In crystallo spectroscopy enables the monitoring of X-ray-induced changes in the spectroscopic signatures of proteins and ligands; however, a comprehensive approach that also considers buffer components is required. Here, we present a mapping of X-ray-induced spectral changes in common crystallization chemicals and mixtures at cryogenic temperature, for which spectroscopic changes can be detected at doses as low as 1 kGy. A transient increase in absorption between 450 and 700 nm is frequently observed, arising from solvated electron absorption. Below 450 nm, several distinct absorption peaks were detected, for example for halides in buffers. In addition, Rayleigh scattering can lead to an increasing loss of photons from the optical path as the wavelength decreases, and thus to a significant elevation of the baseline at shorter wavelengths. In this study, we demonstrate the use of spectroscopic analyses to investigate the chromophoric enzyme intermediate I320 in vitamin B6 biosynthesis. In this case, X-ray-induced spectral changes were attributed to crystallization agents, cryoprotectants and light scattering, thereby excluding intrinsic alterations to the enzyme intermediate under low-dose conditions. Our study highlights the importance of monitoring spectral changes during diffraction data collection to ensure accurate interpretation of the electronic structure of chromophores.
|
May 2026
|
|
|
|
Open Access
Abstract: Serial crystallography relies on the reproducible production of high-density suspensions of microcrystals, yet sample optimization remains a resource-intensive bottleneck. While phase diagrams provide a theoretical framework for controlling crystal size and number, experimental mapping is traditionally hindered by relatively high sample consumption. We present an automated microbatch-under-oil crystallization approach that rapidly maps phase boundaries using only 15–60 µl (∼0.15–3.8 mg) of protein. While this workflow is ideally suited for refining existing hits, it serves as a standalone platform for characterizing the crystallization landscape of new protein targets. The power of this approach lies in the integration of three distinct strategies that exploit the stable chemical environment of microbatch-under-oil. Firstly, we utilize an ingenious diagonal sampling strategy that traverses the phase boundary parallel to the solubility curve by systematically varying protein-to-precipitant ratios, identifying primary nucleation zones with far greater efficiency than traditional orthogonal grids. Secondly, we employ a linked variation of multiple precipitants to reveal morphology-specific regions, such as the rod versus plate transitions crucial for time-resolved experiments. Finally, we incorporate automated seed-stock titration to precisely define the metastable zone, enabling the predictive rescue of nucleation-limited systems. The synergy of these three strategies enables the systematic decoupling of nucleation from growth, providing a rational route to optimize microcrystal density, size and lattice order. Crucially, by eliminating the evaporation-related variables inherent in vapor diffusion, this method ensures that the chemical coordinates identified during screening remain constant during scale-up to larger volumes. This workflow transforms empirical serial crystallography sample preparation into a rational, reproducible and highly efficient process applicable to both the optimization of known conditions and the de novo development of microcrystal suspensions, tailored to the rigorous demands of modern serial diffraction experiments.
|
Mar 2026
|
|
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
|
Hans E.
Pfalzgraf
,
Aditya G.
Rao
,
Kakali
Sen
,
Hannah R.
Adams
,
Marcus
Edwards
,
You
Lu
,
Chin
Yong
,
Sofia
Jaho
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Sam
Horrell
,
James
Beilsten-Edmands
,
Robin L.
Owen
,
Colin R.
Andrew
,
Jonathan A. R.
Worrall
,
Ivo
Tews
,
Adrian J.
Mulholland
,
Michael A.
Hough
,
Thomas W.
Keal
Diamond Proposal Number(s):
[27313]
Open Access
Abstract: Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type haem where the porphyrin is crosslinked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 haem oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460-containing hydroxylamine oxidoreductase utilises a tyrosine cross link to haem and performs similar chemistry. Previous crystal structures of cytochrome P460 from Nitrosomonas europaea (NeP460) clearly show the existence of a single crosslink between the NZ atom of lysine and the haem porphyrin, with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the haem. Here we describe the evidence for a novel double crosslink between lysine and haem in the cytochrome P460 from Methylococcus capsulatus (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this, we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.
|
Aug 2025
|
|
|
|
Open Access
Abstract: Advancements in macromolecular crystallography, driven by improved sources and cryocooling techniques, have enabled the use of increasingly smaller crystals for structure determination, with microfocus beamlines now widely accessible. Initially developed for challenging samples, these techniques have culminated in advanced beamlines such as VMXm. Here, an in vacuo sample environment improves the signal-to-noise ratio in X-ray diffraction experiments, and thus enables the use of submicrometre crystals. The advancement of techniques such as microcrystal electron diffraction (MicroED) for atomic-level insights into charged states and hydrogen positions, along with room-temperature crystallography to observe physiological states via serial crystallography, has driven a resurgence in the use of microcrystals. Reproducibly preparing small crystals, especially from samples that typically yield larger crystals, requires considerable effort, as no one singular approach guarantees optimal crystals for every technique. This review discusses methods for generating such small crystals, including mechanical crushing and batch crystallization with seeding, and evaluates their compatibility with microcrystal data-collection modalities. Additionally, we examine sample-delivery methods, which are crucial for selecting appropriate crystallization strategies. Establishing reliable protocols for sample preparation and delivery opens new avenues for macromolecular crystallography, particularly in the rapidly progressing field of time-resolved crystallography.
|
May 2025
|
|
I23-Long wavelength MX
|
Isabel G.
Elliott
,
Hayden
Fisher
,
H. T. Claude
Chan
,
Tatyana
Inzhelevskaya
,
C. Ian
Mockridge
,
Christine A.
Penfold
,
Patrick J.
Duriez
,
Christian M.
Orr
,
Julie
Herniman
,
Kri T. J.
Müller
,
Jonathan W.
Essex
,
Mark S.
Cragg
,
Ivo
Tews
Diamond Proposal Number(s):
[29835]
Open Access
Abstract: A promising strategy in cancer immunotherapy is activation of immune signalling pathways through antibodies that target co-stimulatory receptors. hIgG2, one of four human antibody isotypes, is known to deliver strong agonistic activity, and modification of hIgG2 hinge disulfides can influence immune-stimulating activity. This was shown for antibodies directed against the hCD40 receptor, where cysteine-to-serine exchange mutations caused changes in antibody conformational flexibility. Here we demonstrate that the principles of increasing agonism by restricting antibody conformation through disulfide modification can be translated to the co-stimulatory receptor h4-1BB, another member of the tumour necrosis factor receptor superfamily. Furthermore, we explore structure-guided design of the anti-hCD40 antibody ChiLob7/4 and show that engineering additional disulfides between opposing F(ab’) arms can elicit conformational restriction, concomitant with enhanced agonism. These results support a mode where subtle increases in rigidity can deliver significant improvements in immunostimulatory activity, thus providing a strategy for the rational design of more powerful antibody therapeutics.
|
Apr 2025
|
|
|
|
Open Access
Abstract: The switch between planktonic and biofilm lifestyle correlates with intracellular concentration of the second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). While bacteria possess cyclase and phosphodiesterase enzymes to catalyse formation or hydrolysis of c-di-GMP, both enzymatic domains often occur in a single protein. It is tacitly assumed that one of the two enzymatic activities is dominant, and that additional domains and protein interactions enable responses to environmental conditions and control activity. Here we report the structure of the phosphodiesterase domain of the membrane protein RbdA (regulator of biofilm dispersal) in a dimeric, activated state and show that phosphodiesterase activity is controlled by the linked cyclase. The phosphodiesterase region around helices α5/α6 forms the dimer interface, providing a rationale for activation, as this region was seen in contact with the cyclase domain in an auto-inhibited structure previously described. Kinetic analysis supports this model, as the activity of the phosphodiesterase alone is lower when linked to the cyclase. Analysis of a computed model of the RbdA periplasmatic domain reveals an all-helical architecture with a large binding pocket that could accommodate putative ligands. Unravelling the regulatory circuits in multi-domain phosphodiesterases like RbdA is important to develop strategies to manipulate or disperse bacterial biofilms.
|
Aug 2024
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[29835]
Open Access
Abstract: The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodelling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific endoglycosidases, developed to enable customisable antibody glycosylation.
|
Apr 2024
|
|
|
|
Jack
Stubbs
,
Theo
Hornsey
,
Niall
Hanrahan
,
Luis Blay
Esteban
,
Rachel
Bolton
,
Martin
Maly
,
Shibom
Basu
,
Julien
Orlans
,
Daniele
De Sanctis
,
Jung-Uk
Shim
,
Patrick D.
Shaw Stewart
,
Allen M.
Orville
,
Ivo
Tews
,
Jonathan
West
Open Access
Abstract: Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.
|
Mar 2024
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Mar 2024
|
|
