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Instruments / Technical Area	Publication Details	Published
	Pr and Pfr structures of plant phytochrome A Soshichiro Nagano , David Von Stetten , Kaoling Guan , Peng-Yuan Chen , Chen Song , Thomas Barends , Manfred S. Weiss , Christian G. Feiler , Katerina Dörner , Iñaki De Diego Martinez , Robin Schubert , Johan Bielecki , Lea Brings , Huijong Han , Konstantin Kharitonov , Chan Kim , Marco Kloos , Jayanath C. P. Koliyadu , Faisal H. M. Koua , Ekaterina Round , Abhisakh Sarma , Tokushi Sato , Christina Schmidt , Joana Valerio , Agnieszka Wrona , Joachim Schulz , Raphael De Wijn , Romain Letrun , Richard Bean , Adrian Mancuso , Karsten Heyne , Jon Hughes Nature Communications 16 DOI: 10.1038/s41467-025-60738-w Open Access Show Abstract ▼ Abstract: Phytochromes are biliprotein photoreceptors widespread amongst microorganisms and ubiquitous in plants where they control developmental processes as diverse as germination, stem elongation and floral induction through the photoconversion of inactive Pr to the Pfr signalling state. Here we report crystal structures of the chromophore-binding module of soybean phytochrome A, including ~2.2 Å XFEL structures of Pr and Pfr at ambient temperature and high resolution cryogenic structures of Pr. In the Pfr structure, the chromophore is exposed to the medium, the D-ring remaining α-facial following the likely clockwise photoflip. The chromophore shifts within its pocket, while its propionate side chains, their partners as well as three neighbouring tyrosines shift radically. Helices near the chromophore show substantial shifts that might represent components of the light signal. These changes reflect those in bacteriophytochromes despite their quite different signalling mechanisms, implying that fundamental aspects of phytochrome photoactivation have been repurposed for photoregulation in the eukaryotic plant.	Jun 2025
I24-Microfocus Macromolecular Crystallography	Multiplexing methods in dynamic protein crystallography Margaret A. Klureza , Yelyzaveta Pulnova , David Von Stetten , Robin L. Owen , Godfrey S. Beddard , Arwen R. Pearson , Briony A. Yorke DOI: 10.1016/bs.mie.2024.10.009 Show Abstract ▼ Abstract: Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.	Oct 2024
I24-Microfocus Macromolecular Crystallography	An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin Jake A. Hill , Yvonne Nyathi , Sam Horrell , David Von Stetten , Danny Axford , Robin L. Owen , Godfrey S. Beddard , Arwen R. Pearson , Helen M. Ginn , Briony Yorke Communications Chemistry 7 DOI: 10.1038/s42004-024-01163-w Diamond Proposal Number(s): [27314] Open Access Show Abstract ▼ 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.	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
	X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease Sebastian Gunther , Patrick Y. A. Reinke , Yaiza Fernández-García , Julia Lieske , Thomas J. Lane , Helen M. Ginn , Faisal H. M. Koua , Christiane Ehrt , Wiebke Ewert , Dominik Oberthuer , Oleksandr Yefanov , Susanne Meier , Kristina Lorenzen , Boris Krichel , Janine-Denise Kopicki , Luca Gelisio , Wolfgang Brehm , Ilona Dunkel , Brandon Seychell , Henry Gieseler , Brenna Norton-Baker , Beatriz Escudero-Pérez , Martin Domaracky , Sofiane Saouane , Alexandra Tolstikova , Thomas A. White , Anna Hänle , Michael Groessler , Holger Fleckenstein , Fabian Trost , Marina Galchenkova , Yaroslav Gevorkov , Chufeng Li , Salah Awel , Ariana Peck , Miriam Barthelmess , Frank Schluenzen , Paulraj Lourdu Xavier , Nadine Werner , Hina Andaleeb , Najeeb Ullah , Sven Falke , Vasundara Srinivasan , Bruno Alves França , Martin Schwinzer , Hévila Brognaro , Cromarte Rogers , Diogo Melo , Joanna J. Zaitseva-Doyle , Juraj Knoska , Gisel E. Peña-Murillo , Aida Rahmani Mashhour , Vincent Hennicke , Pontus Fischer , Johanna Hakanpää , Jan Meyer , Philip Gribbon , Bernhard Ellinger , Maria Kuzikov , Markus Wolf , Andrea R. Beccari , Gleb Bourenkov , David Von Stetten , Guillaume Pompidor , Isabel Bento , Saravanan Panneerselvam , Ivars Karpics , Thomas R. Schneider , Maria Marta Garcia-Alai , Stephan Niebling , Christian Günther , Christina Schmidt , Robin Schubert , Huijong Han , Juliane Boger , Diana C. F. Monteiro , Linlin Zhang , Xinyuanyuan Sun , Jonathan Pletzer-Zelgert , Jan Wollenhaupt , Christian G. Feiler , Manfred S. Weiss , Eike-Christian Schulz , Pedram Mehrabi , Katarina Karničar , Aleksandra Usenik , Jure Loboda , Henning Tidow , Ashwin Chari , Rolf Hilgenfeld , Charlotte Uetrecht , Russell Cox , Andrea Zaliani , Tobias Beck , Matthias Rarey , Stephan Günther , Dusan Turk , Winfried Hinrichs , Henry N. Chapman , Arwen R. Pearson , Christian Betzel , Alke Meents Science DOI: 10.1126/science.abf7945 Open Access Show Abstract ▼ Abstract: The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.	Apr 2021
	Serial femtosecond and serial synchrotron crystallography can yield data of equivalent quality: A systematic comparison P. Mehrabi , R. Bücker , G. Bourenkov , H. M. Ginn , D. Von Stetten , H. M. Müller-Werkmeister , A. Kuo , T. Morizumi , B.t. Eger , W.-L. Ou , S. Oghbaey , A. Sarracini , J. E. Besaw , O. Pare´-Labrosse , S. Meier , H. Schikora , F. Tellkamp , A. Marx , D. A. Sherrell , D. Axford , R. I. Owen , O. P. Ernst , E. F. Pai , E. C. Schulz , R. J. D. Miller Science Advances 7 DOI: 10.1126/sciadv.abf1380 Open Access Show Abstract ▼ Abstract: For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution.	Mar 2021
I02-Macromolecular Crystallography	Direct evidence for a peroxide intermediate and a reactive enzyme–substrate–dioxygen configuration in a cofactor-free oxidase Soi Bui , David Von stetten , Pablo G. Jambrina , Thierry Prangé , Nathalie Colloc'H , Daniele De sanctis , Antoine Royant , Edina Rosta , Roberto A. Steiner Angewandte Chemie International Edition 53 (50), 13710 - 13714 DOI: 10.1002/anie.201405485 PMID: 25314114 Open Access Show Abstract ▼ Abstract: Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Low X-ray doses break specifically the intermediate C5OO(H) bond at 100 K, thus releasing O2 in situ, which is trapped above the substrate radical. The dose-dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick-starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site.	Dec 2014

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