BEGIN:VCALENDAR VERSION:2.0 PRODID:-//CERN//INDICO//EN BEGIN:VEVENT SUMMARY:Fragment Based Active Site Exploration of Polyurethane Degrading E nzymes for Structure-guided Protein Engineering DTSTART;VALUE=DATE-TIME:20250922T174000Z DTEND;VALUE=DATE-TIME:20250922T175000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1866@lindico453.srv.lu.se DESCRIPTION:Speakers: Deniz Bicer (Aarhus University)\nPolyurethane (PU) p lastics are extermyl durable and hard to recycle by convential methods due to\ncrosslinked and branched molecular structure. Recent discoveries of e nzyme that arget carbamate\n(urethane) bond in the PU provide alternative way of PU recycling by means of biocatalytic degradation.\nEnZync center h as been established to discover\, characterize and engineer novel enzymes for PU\ndegradation. So far we have discovered several enzymes by computat ional searches and experimental\nways. The critical steps to establish rat ional basis of protein engineering campaing is the understanding\nof Plast ic-enzyme interactions at molecular level. To pursue structural characteri zation of “PURases”\nwe have two different but complemantry methods to characteirze active site of PURases: i) Fragment\nBased Active site Explo ration (FASE) of PURases by using small molecule fragment libraries and\ns oluble PU analogs and ii) time-resolved serial crystallography by cryocapt uring catalytic intermideate\n(for slow-milisecond kinetics) and ambient t emperature (fast-microsecond) crystallography. We have\ncompleted FASE app roaches for one of our patented enzymes and now we are strating to perform serial\ncrystallography approach with promising preliminary data.\n\nAuth ors: Deniz Bicer\, Laura Rotilio\, Daniel Otzen & Jens Preben Morth\n\nhtt ps://lindico453.srv.lu.se/event/583/contributions/1866/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1866/ END:VEVENT BEGIN:VEVENT SUMMARY:MicroMAX - A beamline with time-resolved macromolecular crystallog raphy capabilities at the MAX IV Laboratory DTSTART;VALUE=DATE-TIME:20250922T173000Z DTEND;VALUE=DATE-TIME:20250922T174000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1865@lindico453.srv.lu.se DESCRIPTION:Speakers: Jie Nan et al (MAX IV Laboratory)\nThe rise of 4th g eneration sources\, including the MAX IV Laboratory 3 GeV ring\, has enabl ed new possibilities to study dynamics using crystallography. The MicroMAX beamline is a new beamline focussed on providing optimal X-ray characteri stics for serial (SSX) and time-resolved (TR-SSX) crystallography at MAX I V [1]. The beamline emphasizes a flexible sample environment for standard and bespoke experimental setups while also supporting high-throughput sing le crystal data collections at the BioMAX beamline which has operated sinc e 2017 [2]. \nThe MicroMAX user program opened in May 2024 and has perform ed experiments with SPINE-based fixed targets\, high-viscosity extrusion a nd microfluidics and single-crystal oscillation data collections. Sample h andling and positioning is supported by the MD3-up micro diffractometer\, Oxford cryojet\, and ISARA automated sample mounting platform (including c rystallization plates). Time resolved techniques are enabled by a nanoseco nd pump laser (210-2600 nm)\, Celerotron X-ray chopper (0\,8-70% duty cycl e) and one of either an Eiger2 X 9M CdTe photon counting hybrid pixel dete ctor or Jungfrau 9M Si integrating hybrid pixel detector (on-loan from PSI ). \nOptical elements allow for a beamline flux from 10^13 photons/s (0.1% bandwidth double crystal monochromator) to more than 10^14 photons/s (1% bandwidth multilayer monochromator) with an optimal 1x1 μm beam focus usi ng beryllium lenses/K-B mirrors. Beamline controls are from within MXCuBE\ , with additional live feedback and CrystFEL autoprocessing pipelines to p rovide immediate feedback and rapid map generation. Sample pre-characteriz ation is supported by an offline laser and spectroscopy lab in the seconda ry experimental hutch and dedicated sample environment and preparation lab s.\nHere we present the current status of MicroMAX beamline and recent dev elopments in sample preparation and data handling under a variety of exper imental contexts. This work emphasizes the technical developments for a hi ghly flexible TR-SSX end station in context of SSX/TR-SSX experiments alre ady being conducted by the MicroMAX user community.\n\nMicroMAX is funded by the Novo Nordisk Foundation under the grant number NNF17CC0030666.\n\n[ 1] Gonzalez\, A.\, Krojer\, T.\, Nan\, J.\, Bjelcic\, M.\, Aggarwal\, S.\, Gorgisyan\, I.\, Milas\, M.\, Eguiraun\, M.\, Casadei\, C.\, Chenchiliyan \, M.\, Jurgilaitis\, A.\, Kroon\, D.\, Ahn\, B.\, Ekstrom\, J. C.\, Aurel ius\, O.\, Lang\, D.\, Ursby\, T. & Thunnissen\, M. M. G. M. (2025). J. Sy nchrotron Rad. 32.\n[2] Shilova\, A.\, Lebrette\, H.\, Aurelius\, O.\, Nan \, J.\, Welin\, M.\, Kovacic\, R.\, Ghosh\, S.\, Safari\, C.\, Friel\, R. J.\, Milas\, M.\, Matej\, Z.\, Högbom\, M.\, Brändén\, G.\, Kloos\, M.\ , Shoeman\, R. L.\, Doak\, B.\, Ursby\, T.\, Håkansson\, M.\, Logan\, D. T. & Mueller U. (2020). J. Synchrotron Rad.\, 27\, 1095.\n\nhttps://lindic o453.srv.lu.se/event/583/contributions/1865/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1865/ END:VEVENT BEGIN:VEVENT SUMMARY:Laser and Spectroscopic Capabilities at MicroMAX DTSTART;VALUE=DATE-TIME:20250922T172000Z DTEND;VALUE=DATE-TIME:20250922T173000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1864@lindico453.srv.lu.se DESCRIPTION:Speakers: Manoop Chenchiliyan (MAX IV Laboratory)\, Sofia M. K apetanaki (MAX IV Laboratory)\nSpectroscopic techniques provide a powerful means of obtaining detailed information on the structural and dynamic pro perties of proteins in solution and in crystallo (proteins and enzymes are active in the crystalline state). Structural data obtained by X-ray cryst allography is strengthened by insights from complementary approaches like UV/vis\, fluorescence and (resonance) Raman measurements.\n\nhttps://lindi co453.srv.lu.se/event/583/contributions/1864/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1864/ END:VEVENT BEGIN:VEVENT SUMMARY:Posters & mingle food (Poster list with abstracts below) DTSTART;VALUE=DATE-TIME:20250922T151500Z DTEND;VALUE=DATE-TIME:20250922T153000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1834@lindico453.srv.lu.se DESCRIPTION:https://lindico453.srv.lu.se/event/583/contributions/1834/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1834/ END:VEVENT BEGIN:VEVENT SUMMARY:Time-resolved serial crystallograhy to capture reaction intermedia tes of a glucuronyl esterase DTSTART;VALUE=DATE-TIME:20250922T154000Z DTEND;VALUE=DATE-TIME:20250922T155000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1833@lindico453.srv.lu.se DESCRIPTION:Speakers: Gabrielle Wehlander (University of Gothenburg)\nGluc uronyl esterases (GEs) from the carbohydrate esterase family 15 (CE15) are involved in degrading lignocellulosic biomass\, by catalyzing the hydroly sis of an esterbond connecting lignin and hemicellulose in the plant cell wall (1). In order to utilize biomass in biorefineries\, efficient methods are needed to separate cellulose\, hemicellulose and lignin. Studying GEs to better understand their reaction mechanism can aid in improving existi ng biological preteatment methods used in biorefineries to be able to make better use of this renewable energy source. The bacterial GE from Opitutu s terrae (OtCE15A) has previously been structurally determined at cryo-tem perature and a reaction mechanism for the acylation and deacylation reacti ons has been proposed (2\,3). Various glucuronate- and galacturonate ester s have been used as model substrates for the lignin-hemicellulose linkage\ , and the substrates have been soaked into the crystals. However\, attempt s to capture the binding of the substrates prior to hydrolysis of the este r bond have so far been unsuccessful\, but a covalent reaction intermediat e has been obtained using enzymes with mutations at the catalytic site. In attempts to capture the binding of substrates prior to hydrolysis and to determine reaction intermediates\, we have collected serial synchrotron X- ray crystallography (SSX) data at BioMAX (MAX IV\, Lund)\, and conducted i nitial time-resolved SSX experiment at P14.EH2 (T-REXX of PETRA III\, Hamb urg). We have obtained high resolution (1.7Å) SSX data of OtCE15A at BioM AX and observed binding of the cleaved substrate of benzyl glucuronoate af ter a soaking time of 5 minutes. For time-resolved SSX experiments at T-RE XX\, we have tested and are planning to use the hit-and-return (HARE) meth od (4).\n\nReferences\n1. Larsbrink\, Johan\, and Leila Lo Leggio. "Glucur onoyl esterases–enzymes to decouple lignin and carbohydrates and enable better utilization of renewable plant biomass." Essays in Biochemistry 67. 3 (2023): 493-503.\n2. Mazurkewich\, Scott\, et al. "Structural and bioche mical studies of the glucuronoyl esterase OtCE15A illuminate its interacti on with lignocellulosic components." Journal of Biological Chemistry 294.5 2 (2019): 19978-19987.\n3. Zong\, Zhiyou\, et al. "Mechanism and biomass a ssociation of glucuronoyl esterase: an α/β hydrolase with potential in b iomass conversion." Nature Communications 13.1 (2022): 1449.\n4. Schulz\, Eike C.\, et al. "The hit-and-return system enables efficient time-resolve d serial synchrotron crystallography." Nature methods 15.11 (2018): 901-90 4.\n\nAuthors: Gabrielle Wehlander\, Josefin Ridaeus\, Scott Mazurkewich\, Leila Lo Leggio\, Johan Larsbrink\, Gisela Brändén\n\nhttps://lindico45 3.srv.lu.se/event/583/contributions/1833/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1833/ END:VEVENT BEGIN:VEVENT SUMMARY:Standard Sample Preparation and Characterization for Serial Crysta llography DTSTART;VALUE=DATE-TIME:20250922T171000Z DTEND;VALUE=DATE-TIME:20250922T172000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1842@lindico453.srv.lu.se DESCRIPTION:Speakers: Christina Schmidt (European XFEL)\nSerial crystallog raphy (SX) has been a revolutionary technique in structural biology for mo re than a decade\, providing insights into the structures and dynamics of biomolecules at room temperature. Using intense ultra-short X-ray pulses\, SX made the collection of diffraction data from micron-sized crystals pos sible\, avoiding radiation damage and allowing for the capture of transien t states and intermediates in biological reactions. The success of SX expe riments heavily relies on the quality of the sample.\nStandard samples in SX research provide critical roles. First\, the commissioning of new beaml ine devices needs well-characterized samples. By providing consistent and reproducible diffraction patterns\, the standard samples help in the calib ration of new devices and verification of their performance. Secondly\, th ey are required for the validation of experimental setups\, ensuring that all components\, from sample delivery systems to data acquisition software \, function correctly.\nIn this study\, we present detailed protocols for the preparation of lysozyme\, myoglobin\, iq-mEmerald\, and photoactive ye llow protein (PYP) crystals. These proteins were selected as standard samp les due to their robust crystallization properties and suitability for a w ide range of SX experiments. Through the optimization of existing protocol s\, we achieved high-quality crystal samples with improved yield\, specifi cally for SX applications.\n\n\nAuthors: Christina Schmidt\, Huijong Han\n \nhttps://lindico453.srv.lu.se/event/583/contributions/1842/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1842/ END:VEVENT BEGIN:VEVENT SUMMARY:Time and dose resolved crystallography to control and capture redo x states in heme peroxidases DTSTART;VALUE=DATE-TIME:20250922T170000Z DTEND;VALUE=DATE-TIME:20250922T171000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1841@lindico453.srv.lu.se DESCRIPTION:Speakers: Michael Hough (Diamond Light Source)\nMetalloenzymes containing heme centres catalyse a wide range of reactions critical to li fe. Understanding the structure and electronic states of the heme centre a cross multiple functionally relevant states is essential to understand mec hanism. I will describe work using time resolved serial crystallography as well as the use of X-ray dose for the manipulation of heme iron oxidation states in dye decolourising peroxidases [1] using multiple\, complementar y\, serial crystallography and single-crystal spectroscopic approaches.\n\ nFixed target drop-on-chip\, tape drive droplet on demand and correlated s pectroscopies allow the formation of high valent Fe(IV) states to be chara cterised. X-ray Pump Probe serial femtosecond crystallography (SFX) togeth er with dose-resolved serial synchrotron crystallography (SSX) allowed the peroxidases to be driven between multiple iron oxidation states that can be spectroscopically validated. Intriguingly\, the formation and dose resp onse of the Fe(IV)-O state is highly variable between the chemically ident ical heme groups of the homo-oligomeric proteins highlighting the importan ce of understanding the effect of the crystalline lattice on observed chan ges in time- and dose-resolved crystallography experiments.\n\nLucic\, M. et al (2021) Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial d ye-decolorizing peroxidases. JBIC 27 (7)\, 743-761.\n\n\nCo-authors: Robin Owen\, Jonathan Worrall\, Marina Rozman\, Lewis Williams\, Danny Axford\, Allen Orville\, Pierre Aller\, Jan Kern\, Jos Kamps\n\nhttps://lindico453 .srv.lu.se/event/583/contributions/1841/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1841/ END:VEVENT BEGIN:VEVENT SUMMARY:Experimental estimation of copper-ligand length precision in a mod el fungal LPMO under redox cycling and saccharide binding DTSTART;VALUE=DATE-TIME:20250922T165000Z DTEND;VALUE=DATE-TIME:20250922T170000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1840@lindico453.srv.lu.se DESCRIPTION:Speakers: Zhiyu Huang (University of Copenhagen)\nLytic polysa ccharide monooxygenases (LPMOs) are copper-dependent enzymes that degrade polysaccharides oxidatively\, with applications in second-generation bioet hanol production and as virulence factors in certain pathogens [1] [2]. Th ey have been reclassified within the CAZy database into auxiliary activity families AA9–AA11 and AA13–AA17 [3]\, and their active-site histidine brace is highly conserved. The catalytic mechanism of LPMOs is complex\, with the priming reaction step requiring the reduction of Cu(II) to Cu(I). Since the geometric changes associated with redox cycling—whether chemi cally induced or triggered by photoreduction—can be subtle\, the accurat e determination of bond lengths and angles is essential [4]. In this study \, LsAA9A from Lentinus similis was used as a model system under various e xperimental conditions. Analysis of the LsAA9A_Ec and LsAA9A_Ec_Cell3 stru ctures collected under low-dose conditions showed that\, compared with oth er Cu-coordination distances\, only the Tyr–Cu bond exhibited a statisti cally significant change (p = 0.00094 in two tailed t-test). These finding s confirm that saccharide substrate binding consistently shortens the Tyr –Cu distance in LsAA9A_Ec in the Cu²⁺ state\, with a measured reducti on of 0.21 Å. Furthermore\, experiments on LsAA9A_Ec_Asc and LsAA9A_Ec_As c_Cell3\, conducted under both low- and high-dose conditions\, where Cu² ⁺ can be reduced to Cu⁺ by X-ray exposure\, as well as at room tempera ture\, further probed structural responses to varying redox and experiment al regimes. These findings advance the mechanistic understanding of LPMOs and offer a framework for probing subtle geometric changes in metalloenzym es.\n\nAuthors: Zhiyu Huang\, Jie Nan\, Monika Bjelcic\, Leila Lo Leggio\n \nhttps://lindico453.srv.lu.se/event/583/contributions/1840/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1840/ END:VEVENT BEGIN:VEVENT SUMMARY:Serial X – Simple Solutions for Serial Synchrotron Crystallograp hy DTSTART;VALUE=DATE-TIME:20250922T164000Z DTEND;VALUE=DATE-TIME:20250922T165000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1844@lindico453.srv.lu.se DESCRIPTION:Speakers: Yanyan Chen (Serial X AB)\nSerial X aims to expand o f the use of serial crystallography across the academic and industrial lif e-science community by introducing low cost\, flexible and easy to use sol utions to the problem of sample delivery in serial crystallography experim ents at synchrotron radiation facilities. These solutions were invented in ProtonPump\, the ERC Advanced Grant awarded to Richard Neutze\, which app lies time-resolved serial crystallography to observe structural changes in the enzyme cytochrome c oxidase. Serial crystallography is going through a period of rapid development and the number of scientific users of this m ethod has the potential to grow by up-to two orders of magnitude. This pot ential will never be realized if the current lack of standardization in sa mple delivery continues\, which is prohibitively expensive and is often un reliable. Serial-X will solve this problem by developing and bringing to m arket standardized\, low-cost\, flexible and easy-to-use solutions for sam ple delivery in serial crystallography studies at synchrotron radiation so urces. With the support of ERC proof-of-concept grant to Richad Neutze\, S erial X has developed low-cost products supporting both flow cell1 and fix ed target2 approaches to serial crystallography that are mounted upon stan dard magnets used at all conventional macromolecular X-ray crystallography synchrotron-based beamlines. This will remove the greatest obstacle curre ntly preventing scientists from using serial crystallography for their own research or for structure based drug-design within a pharmaceutical drug discovery context.\n\n\nAuthors: Yanyan Chen\, Swagatha Ghosh\, Gisela Br ändén\, Richard Neutze\n\nhttps://lindico453.srv.lu.se/event/583/contrib utions/1844/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1844/ END:VEVENT BEGIN:VEVENT SUMMARY:Reducing data volume with X-ray Laue diffraction DTSTART;VALUE=DATE-TIME:20250922T163000Z DTEND;VALUE=DATE-TIME:20250922T164000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1839@lindico453.srv.lu.se DESCRIPTION:Speakers: Kah Chee Pow (Spallation Neutron Source Science Cent er)\nThe X-ray Laue diffraction captures the crystal diffraction from whit e beam and can contain significant amount of structural information in com parison to monochromatic diffraction. In time-resolved study\, the serial femtosecond crystallography (SFX) and serial synchrotron crystallography ( SSX) are the mainstream approaches with the caveat of generating large dat a volume. Laue diffraction has great potential in mitigating the data volu me challenge and has recently regain the interest by the community. Here\, we share the preliminary works conducted at BL03HB Laue Micro-diffraction Beamline at Shanghai Synchrotron Radiation Facility (SSRF) to compare wit h conventional method. We crystallized an apo stilbene synthase (STS) with inherent loop conformation duality and collected diffraction data via (i) single-crystal\, cryogenic\, rotation approach with monochromatic beam\, (ii) single-crystal\, ambient\, helical approach with white beam\, and (ii i) multi-crystal\, ambient\, single-frame approach with white beam. Compar e to monochromatic diffraction\, Laue diffraction dataset show increased c ontent of structural information per image but with poor statistical metri cs upon merging. Nevertheless\, the solved structure from Laue diffraction dataset can resolve the duality of the loop conformation and reveal the l oop conformational preference at productive state at ambient temperature. In short\, Laue diffraction is a promising approach to explore in mitigati ng the data volume challenge.\n\nAuthor: Kah Chee Pow\nCo-author: Quan Hao \n\nhttps://lindico453.srv.lu.se/event/583/contributions/1839/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1839/ END:VEVENT BEGIN:VEVENT SUMMARY:Structural studies of the human drug-metabolising protein CYP3A4 DTSTART;VALUE=DATE-TIME:20250922T161000Z DTEND;VALUE=DATE-TIME:20250922T162000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1838@lindico453.srv.lu.se DESCRIPTION:Speakers: Johan Glerup (University of Gothenburg)\nA highly fl exible protein with an active site that changes its volume to fit a wide v ariety of ligands. A lid made of loops changes conformation based on ligan d-size. Inhibition of this enzyme stops metabolism of drugs. This is an au tomatic disqualification of a drug candidate. Room temperature SSX shows b etter definition of some flexible loops\, even at worse resolution. SSX da ta collection at tens of kilo Gray produces a similar active-site to our X FEL structure. I present an internal distance matrix analysis of a subset of PDB CYP3A4 structures to determine that crystal form\, resolution and t o some extent ligand-size dominates the clustering of global similarity wi th little difference caused by temperature. The protein crystalises as a m onomer in the ASU but SAX\, cryo-EM and SEC-MALS shows a homo-tetramer in solution bringing it into the perfect size range for cryo-EM. I present in itial data for the volume of the tetramer solved through single particle a nalysis at SciLifeLab Solna.\n\n\nCo-authors: Owens Uwangue\, Gisela Brand en\, Monika Bjelcic\n\nhttps://lindico453.srv.lu.se/event/583/contribution s/1838/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1838/ END:VEVENT BEGIN:VEVENT SUMMARY:Development of new data processing methods for serial time-resolve d crystallography DTSTART;VALUE=DATE-TIME:20250922T160000Z DTEND;VALUE=DATE-TIME:20250922T161000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1837@lindico453.srv.lu.se DESCRIPTION:Speakers: Rachel Tang (Diamond Light Source)\nTo obtain the hi ghest quality electron density maps\, from a time-resolved serial crystall ography experiment\, it is crucial to accurately group the observed intens ities according to the structure of the protein that generated those inten sities. In an ideal world\, each unique protein crystal structure would gi ve rise to a set of well defined intensities\, which would enable the inte nsities to be grouped easily. Unfortunately\, due to many real world limit ations\, this is not the case: 1) The crystal is an average of many protei n structures 2) Experimental effects such as unequal soaking\, unequal las er/ x-ray exposure etc. 3) A range of crystal sizes causing inconsistent i ntensities which require rescaling. These effects give rise to uncertainty in the observed intensity values. Furthermore\, only a minor proportion o f hkls are affected by the change in protein structure\, and of those hkls \, the intensity changes are often subtle.\nDespite all the challenges\, a statistically rigorous approach is required to accurately group observed intensities according to the structure of the protein that generated those intensities. One approach utilises Bayesian statistics\, a probability ba sed approached used in many areas such as weather forecasting\, econometri cs and natural language processing. In principle\, some Bayesian methods s uch as naive Bayes and maximum likelihood estimator is able to assess the probability a set of intensities were derived from a protein structure. In practice\, the intensities have high uncertainty values and there are man y nuances as to how this data processing pipeline should be set up\, somet imes sacrificing flexibility for increased confidence in the results.\n\nC o-authors: James Beilsten-Edmands\, Mike Hough\, Graeme Winter\n\nhttps:// lindico453.srv.lu.se/event/583/contributions/1837/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1837/ END:VEVENT BEGIN:VEVENT SUMMARY:Comprehensive Support for Serial Crystallography at the European X FEL DTSTART;VALUE=DATE-TIME:20250922T155000Z DTEND;VALUE=DATE-TIME:20250922T160000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1836@lindico453.srv.lu.se DESCRIPTION:Speakers: Huijong Han (European XFEL)\nSerial crystallography has opened new possibilities for determining the structures of biological macromolecules using microcrystals. At the European XFEL\, the Sample Envi ronment and Characterization (SEC) group\, operating the XBI biology labor atory\, provides essential support for users at every stage of the serial crystallography workflow\, from initial sample preparation to advanced cha racterization and sample delivery.\nThe XBI laboratory offers state-of-the -art facilities where users can prepare and characterize their samples bef ore experiments. Through hands-on support and guidance\, we help users opt imizing their samples to make efficient use of valuable beamtime. The SEC group provides sample delivery methods and supports users in optimizing th eir own systems.\nBy combining expertise in sample delivery and advanced c haracterization\, the SEC group at European XFEL supports the structural b iology community to achieve innovative results in serial crystallography. Our ongoing developments in standard samples\, support infrastructure\, an d efficient sample delivery methods continue to lower access barriers\, fo ster collaboration\, and broaden the access to serial crystallography.\n\n https://lindico453.srv.lu.se/event/583/contributions/1836/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1836/ END:VEVENT BEGIN:VEVENT SUMMARY:In crystallo study of the reaction mechanism in a family B DNA pol ymerase DTSTART;VALUE=DATE-TIME:20250922T153000Z DTEND;VALUE=DATE-TIME:20250922T154000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1835@lindico453.srv.lu.se DESCRIPTION:Speakers: Vimal Parkash (Umeå University)\nReaction intermedi ates during DNA synthesis have been studied in detail using time-resolved\ nX-ray crystallography for translesion and repair DNA polymerases. Contrar y to the originally\nproposed two-metal-ion mechanism\, a third metal ion was identified between the finger\ndomain and the α- and β-phosphates of the incoming nucleotide. This third metal ion was\nsuggested to either pa rticipate in catalysis or stabilize product formation. To investigate this \nfurther in a replicative polymerase\, we conducted time-resolved X-ray c rystallography with\nDNA Polymerase epsilon\, which synthesizes DNA at a m uch faster rate—10x to 100x higher\nthan family Y and X polymerases. Sur prisingly\, no metal ion was observed between the finger\ndomain and the α- and β-phosphates of the incoming nucleotide in any of the solved stru ctures\nwith Pol epsilon. Instead\, our biochemical and structural data su pport the original two-metal\nmechanism. In addition\, we discovered that the 3’-OH group releases a proton\, which is\nchanneled via structural w aters to a basic residue in the Palm domain. After forming a new\nbond wit h the incoming nucleotide\, an acidic residue in the finger domain protona tes the\nreleased pyrophosphate\, stabilizing the product. In summary\, it seems that metal A’s role is\nto lower the pKa of the 3’-OH group\, f ollowed by specific residues in Pol epsilon donating or\nreceiving a proto n to catalyze this acid-base reaction.\n\nCo-author: Erik Johansson\n\nhtt ps://lindico453.srv.lu.se/event/583/contributions/1835/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1835/ END:VEVENT BEGIN:VEVENT SUMMARY:Scientific opportunities for Serial Crystallography at ALBA synchr otron DTSTART;VALUE=DATE-TIME:20250922T162000Z DTEND;VALUE=DATE-TIME:20250922T163000Z DTSTAMP;VALUE=DATE-TIME:20260525T143029Z UID:indico-contribution-214-1843@lindico453.srv.lu.se DESCRIPTION:Speakers: Isidro Crespo (ALBA-CELLS)\, Xavi Carpena i Vilella (ALBA-CELLS)\nThe ALBA Synchrotron\, the 3 GeV light source in Southern Eu rope\, is preparing for its upgrade to a 4th generation storage ring (ALBA II) together with the construction of 3 beamlines and major upgrades on t he existing ones\, early next decade. Within this framework\, serial and t ime-resolved macromolecular crystallography (SSX and TR-MX) are identified as strategic growth areas.\nBL13-XALOC\, in operation since 2012 [1]\, ha s been the workhorse MX beamline at ALBA\, delivering photon fluxes up to 2.5 × 10¹² ph/s with beam sizes from 50 × 7 μm² to 300 × 100 μm² over 5.2–22 keV. The beamline has undergone an upgrade process including a Pilatus-3X 6M (100Hz) detector and a new automated sample changer. Besi des\, the beamline is equipped with a high viscosity extrusion injector fo r SSX experiments. The transition from single crystal oscillation MX to SS X is highly simplified thanks a three-axis motorized stage and sample extr usion is facilitated thanks to a PID pressure control system. Proof-of-con cept SSX experiments with test proteins have demonstrated compatibility wi th room-temperature data collection and negligible radiation damage [2]. P ump–probe TR-MX experiments using visible-light activation have also bee n successfully performed\, establishing a baseline for further development s [3].\nA second MX beamline\, BL06-XAIRA\, started regular user operation in June 2025\, offering a highly stable microfocus beam of 3 × 1 μm² a nd photon energy range of <4.0–14 keV. Equipped with a <60nm runout diff ractometer\, an EIGER2 XE 9M and a dual Channel-Cut/multilayer monochromat or [4]\, it enables high-flux\, high-stability microcrystallography. This equipment\, combined with the implementation in the following months of a fast (up to 750 mm/s) SSX stage\, designed for chips up to 60x40 mm will e nable fixed-target SSX experiments at higher time resolution. Background r eduction and long wavelength experiments are possible due to the recircula ted He environment enclosing the entire end station.\nThe ALBA II upgrade plan foresees substantial investment in SSX/TR-MX capabilities for both be amlines. For XALOC\, this includes hybrid tape-drive systems for tunable s oaking times\, and microfluidic injectors. XAIRA will focus on microfluidi c chips\, acoustic droplet ejection\, and conveyor-belt systems for contin uous fresh sample delivery. A pump-probe set up will be developed for both beamlines to allow for μs–ms TR-MX\, including choppers\, tunable lase rs\, and synchronization systems.\n[1] Juanhuix\, J.\, Gil-Ortiz\, F.\, Cu ní\, G.\, Colldelram\, C.\, Nicolas\, J.\, Lidon\, J.\, Boter\, E.\, Ruge t\, C.\, Ferrer S. & Benach\, J. (2014) Synchrotron Radiat. 21\, 679-689.\ n[2] Martin-Garcia J.M.\, Botha S.\, Hu H.\, Jernigan R.\, Castellví A.\, Lisova S.\, Gil-Ortiz F.\, Calisto B.\, Crespo I.\, Roy-Chowdhury S.\, Gr ieco A.\, Ketawala G.\, Weierstall U.\, Spence J.\, Fromme P.\, Zatsepin N .\, Boer D.R. & Carpena X. (2022) J. Synchrotron Radiat. 29(3): 896-\n[3] Kovalev K\, et al\, Nat Commun. 2020 May 1\;11(1):2137.\n[4] N. González et al.\, Proc. 12th Int. Conf. Mech. Eng. Design Synchrotron Radiat. Equip . Instrum. (MEDSI'23)\, Beijing\, China\, Nov. 2023\, pp.~5-9. doi:10.1842 9/JACoW-MEDSI2023-TUOAM04\n\n\nAuthors:\nXAVI CARPENA I VILELLA\, ISIDRO C RESPO\nCo-authors:\nDAMIÀ GARRIGA\, FERNANDO GIL-ORTIZ\, NAHIKARI GONZÁL EZ\, ALEIX TARRÉS\, ALBERT MIRET\, BERNAT MOLAS\, RICARDO VALCÁRCEL\, AL EJANDRO ENRIQUE\, CARLES COLLDELRAM\, IGORS ŠICS\, JOSÉ MARÍA ÁLVAREZ\ , MARCOS QUISPE\, JULIÁN ALBERTO GARCÍA\, ROELAND BOER\, JUDITH JUANHUIX \n\nhttps://lindico453.srv.lu.se/event/583/contributions/1843/ LOCATION:LINXS at The Loop URL:https://lindico453.srv.lu.se/event/583/contributions/1843/ END:VEVENT END:VCALENDAR