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25 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
	Small but mighty: the power of microcrystals in structural biology Courtney J. Tremlett , Jack Stubbs , William S. Stuart , Patrick D. Shaw Stewart , Jonathan West , Allen M. Orville , Ivo Tews , Nicholas J. Harmer Iucrj 12 DOI: 10.1107/S2052252525001484 Open Access Show Abstract ▼ 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	Structure-guided disulfide engineering restricts antibody conformation to elicit TNFR agonism 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 Nature Communications 16 DOI: 10.1038/s41467-025-58773-8 Diamond Proposal Number(s): [29835] Open Access Show Abstract ▼ 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
	Control of phosphodiesterase activity in the regulator of biofilm dispersal RbdA from Pseudomonas aeruginosa Charlotte Cordery , Jack Craddock , Martin Maly , Kieran Basavaraja , Jeremy S. Webb , Martin A. Walsh , Ivo Tews Rsc Chemical Biology 7 DOI: 10.1039/D4CB00113C Open Access Show Abstract ▼ 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	The IgG-specific endoglycosidases EndoS and EndoS2 are distinguished by conformation and antibody recognition Abigail S. L. Sudol , Max Crispin , Ivo Tews Journal Of Biological Chemistry 11 DOI: 10.1016/j.jbc.2024.107245 Diamond Proposal Number(s): [29835] Open Access Show Abstract ▼ 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
I24-Microfocus Macromolecular Crystallography	A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus 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 Proceedings Of The National Academy Of Sciences 121 DOI: 10.1073/pnas.2308478121 Diamond Proposal Number(s): [15722, 14493, 23570] Open Access Show Abstract ▼ 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
	Droplet microfluidics for time-resolved serial crystallography 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 Iucrj 11, 237 - 248 DOI: 10.1107/S2052252524001799 Open Access Show Abstract ▼ 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
	The CCP 4 suite: integrative software for macromolecular crystallography Jon Agirre , Mihaela Atanasova , Haroldas Bagdonas , Charles B. Ballard , Arnaud Basle , James Beilsten-Edmands , Rafael J. Borges , David G. Brown , J. Javier Burgos-Marmol , John M. Berrisford , Paul S. Bond , Iracema Caballero , Lucrezia Catapano , Grzegorz Chojnowski , Atlanta G. Cook , Kevin D. Cowtan , Tristan I. Croll , Judit É. Debreczeni , Nicholas E. Devenish , Eleanor J. Dodson , Tarik R. Drevon , Paul Emsley , Gwyndaf Evans , Phil R. Evans , Maria Fando , James Foadi , Luis Fuentes-Montero , Elspeth F. Garman , Markus Gerstel , Richard J. Gildea , Kaushik Hatti , Maarten L. Hekkelman , Philipp Heuser , Soon Wen Hoh , Michael A. Hough , Huw T. Jenkins , Elisabet Jiménez , Robbie P. Joosten , Ronan M. Keegan , Nicholas Keep , Eugene B. Krissinel , Petr Kolenko , Oleg Kovalevskiy , Victor S. Lamzin , David M. Lawson , Andrey Lebedev , Andrew G. W. Leslie , Bernhard Lohkamp , Fei Long , Martin Maly , Airlie Mccoy , Stuart J. Mcnicholas , Ana Medina , Claudia Millán , James W. Murray , Garib N. Murshudov , Robert A. Nicholls , Martin E. M. Noble , Robert Oeffner , Navraj S. Pannu , James M. Parkhurst , Nicholas Pearce , Joana Pereira , Anastassis Perrakis , Harold R. Powell , Randy J. Read , Daniel J. Rigden , William Rochira , Massimo Sammito , Filomeno Sanchez Rodriguez , George M. Sheldrick , Kathryn L. Shelley , Felix Simkovic , Adam J. Simpkin , Pavol Skubak , Egor Sobolev , Roberto A. Steiner , Kyle Stevenson , Ivo Tews , Jens M. H. Thomas , Andrea Thorn , Josep Triviño Valls , Ville Uski , Isabel Uson , Alexei Vagin , Sameer Velankar , Melanie Vollmar , Helen Walden , David Waterman , Keith S. Wilson , Martyn Winn , Graeme Winter , Marcin Wojdyr , Keitaro Yamashita Acta Crystallographica Section D Structural Biology 79, 449 - 461 DOI: 10.1107/S2059798323003595 Open Access Show Abstract ▼ Abstract: The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.	Jun 2023
	Reducing affinity as a strategy to boost immunomodulatory antibody agonism Xiaojie Yu , Christian M. Orr , H. T. Claude Chan , Sonya James , Christine A. Penfold , Jinny Kim , Tatyana Inzhelevskaya , C. Ian Mockridge , Kerry L. Cox , Jonathan W. Essex , Ivo Tews , Martin J. Glennie , Mark S. Cragg Nature 102 DOI: 10.1038/s41586-022-05673-2 Show Abstract ▼ Abstract: Antibody responses during infection and vaccination typically undergo affinity maturation to achieve high-affinity binding for efficient neutralization of pathogens1,2. Similarly, high affinity is routinely the goal for therapeutic antibody generation. However, in contrast to naturally occurring or direct-targeting therapeutic antibodies, immunomodulatory antibodies, which are designed to modulate receptor signalling, have not been widely examined for their affinity–function relationship. Here we examine three separate immunologically important receptors spanning two receptor superfamilies: CD40, 4-1BB and PD-1. We show that low rather than high affinity delivers greater activity through increased clustering. This approach delivered higher immune cell activation, in vivo T cell expansion and antitumour activity in the case of CD40. Moreover, an inert anti-4-1BB monoclonal antibody was transformed into an agonist. Low-affinity variants of the clinically important antagonistic anti-PD-1 monoclonal antibody nivolumab also mediated more potent signalling and affected T cell activation. These findings reveal a new paradigm for augmenting agonism across diverse receptor families and shed light on the mechanism of antibody-mediated receptor signalling. Such affinity engineering offers a rational, efficient and highly tuneable solution to deliver antibody-mediated receptor activity across a range of potencies suitable for translation to the treatment of human disease.	Feb 2023
I03-Macromolecular Crystallography	Extensive substrate recognition by the streptococcal antibody-degrading enzymes IdeS and EndoS Abigail S. L. Sudol , John Butler , Dylan P. Ivory , Ivo Tews , Max Crispin Nature Communications 13 DOI: 10.1038/s41467-022-35340-z Diamond Proposal Number(s): [29835] Open Access Show Abstract ▼ Abstract: Enzymatic cleavage of IgG antibodies is a common strategy used by pathogenic bacteria to ablate immune effector function. The Streptococcus pyogenes bacterium secretes the protease IdeS and the glycosidase EndoS, which specifically catalyse cleavage and deglycosylation of human IgG, respectively. IdeS has received clinical approval for kidney transplantation in hypersensitised individuals, while EndoS has found application in engineering antibody glycosylation. We present crystal structures of both enzymes in complex with their IgG1 Fc substrate, which was achieved using Fc engineering to disfavour preferential Fc crystallisation. The IdeS protease displays extensive Fc recognition and encases the antibody hinge. Conversely, the glycan hydrolase domain in EndoS traps the Fc glycan in a “flipped-out” conformation, while additional recognition of the Fc peptide is driven by the so-called carbohydrate binding module. In this work, we reveal the molecular basis of antibody recognition by bacterial enzymes, providing a template for the development of next-generation enzymes.	Dec 2022
I24-Microfocus Macromolecular Crystallography	Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature Tadeo Moreno-Chicano , Leiah M. Carey , Danny Axford , John H. Beale , R. Bruce Doak , Helen M. E. Duyvesteyn , Ali Ebrahim , Robert W. Henning , Diana C. F. Monteiro , Dean A. Myles , Shigeki Owada , Darren A. Sherrell , Megan L. Straw , Vukica Šrajer , Hiroshi Sugimoto , Kensuke Tono , Takehiko Tosha , Ivo Tews , Martin Trebbin , Richard W. Strange , Kevin L. Weiss , Jonathan A. R. Worrall , Flora Meilleur , Robin L. Owen , Reza A. Ghiladi , Michael A. Hough Iucrj 9 DOI: 10.1107/S2052252522006418 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.	Sep 2022

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