Scientific achievements through serial crystallography

22 Sept 2025, 11:00 → 24 Sept 2025, 14:00 Europe/Stockholm

LINXS at The Loop

Welcome to a LINXS-MAX IV symposium

Read more about the event here

When: 22–24 September 2025

Where: LINXS, The Loop, Rydbergs torg 4, Lund, Sweden

Organisers: LINXS, MAX IV

Fee: A registration fee will be charged for the event. Regular 500 SEK, students/postdocs 250 SEK. The fee covers  lunch and coffee breaks on both days and the conference dinner at Kulturens museum on the 23rd September. Participants are expected to arrange for their own travel and stay.

You may find links for hotels/guest houses here: https://visitlund.se/en/accommodation-and-travel

Registration Deadline:  1st September, 2025

Call for abstracts for Poster/ short talks: If you are interested in presenting posters or short talks (15 min), please submit an abstract below.  

Deadline for abstract submission: 28 August, 2025

For practical questions, please contact josefin.martell@linxs.lu.se or isha.raj@maxiv.lu.se

If you don’t receive a registration confirmation please check your spam mail.

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Monday 22 September

11:00 → 13:00 Arrival: Registration & Lunch

11:00 Registration

12:00 Lunch

13:00 → 17:15 Monday: Afternoon

13:00 Welcome (Science Director Joachim Schnadt & MicroMAX Spokesperson Richard Neutze)

Speakers: Prof. Joachim Schnadt (MAX IV Laboratory), Prof. Richard Neutze (University of Gothenburg)

13:10 Some questions can only be answered by time: how time-resolved crystallography reshapes structural biology

Cryo-crystallography was the driving force of structural biology, literally deciphering the structures of life. The integrated knowledge has informed powerful machine learning technologies to not only solve the folding problem but to imagine new proteins, some of which are even functional. However, protein function is often dominated by small kinetic barriers that are not easily predicted. Time-resolved crystallography has set out to answer new questions about protein function and to resolve atomic motion on the femtosecond scale and kinetic intermediates ranging from femtoseconds to seconds - hopefully culminating in the ability to design molecular machines one day. The talk aims to embed some of our research into this greater context.

Speaker: Tobias Weinert (Paul Scherrer Institute)

13:40 The potential for serial crystallography in drug discovery

Andreas Dunge (1,2), Gabrielle Wehlander (2), Gisela Brändén(2) and Helena Käck(1)
1 Protein Sciences, Structure and Biophysics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 2 Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-405 30 Gothenburg, Sweden.

Structure-based drug design has played a pivotal role in pharmaceutical discovery for over thirty years, leading to the development of numerous approved therapeutics. Protein crystallography, relying on data collection from large single crystals held at cryo temperature, provides a highly optimised and effective workflow for generating structures of complexes between compounds and their target protein. Still, this method requires manual manipulation of thousands of crystals per year for a company such as AstraZeneca.
Serial crystallography presents an attractive alternative, offering potential improvements in throughput and automation by circumventing labor-intensive crystal harvesting and facilitating streamlined sample preparation. Additionally, the feasibility of room-temperature data collection can reduce the risk of structural artefacts introduced by cryo-cooling. Nonetheless, the adoption of serial crystallography in early drug discovery remains challenging due to constraints in speed, protein and compound availability, and workflow optimization.
In this study, we demonstrate the development and implementation of an optimized serial crystallography workflow tailored for drug discovery applications. Our approach enables the high-throughput screening of 384 fragments, addressing some key limitations while paving the way for broader application of serial crystallography in pharmaceutical research.

Speaker: Helena Käck

14:10 Coffee break

14:40 From Static to Dynamic: Evolving Methods in Protein Structure Determination

(representing the entire SSRL-SMB team)
Structural Molecular Biology (SMB), Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, Stanford University, Menlo Park, United States of America

Structural biologists are undertaking increasingly challenging projects including the study of membrane proteins and complex multi-component machines. Structural investigations are also transitioning beyond solving a single static structure, to the application of a series of sequential structural snapshots to provide details of the atomic positions and motions that define the relationships involved in molecular recognition, transition state stabilization, and other aspects of the biocatalytic process. The success of these experiments requires careful optimization of samples and experimental setups, often involving multiple experiments at the laboratory bench and the beamline, where automation serves as an enabling technology to efficiently deliver multiple crystals and meet stringent timing requirements.
Developments at SSRL and LCLS-MFX will be presented that tackle challenges involved in the study of metalloenzymes, the use of small and radiation-sensitive crystals, and to perform time-resolved crystallography. To facilitate the handling and optimization of delicate crystals, new in situ crystallization and remote data collection schemes have been released that avoid direct manipulation of crystals, support robotic sample exchange, and allow full rotational access of the sample in a controlled humidity environment. By simplifying crystal handling and transport at near-physiological temperatures, these technologies remove barriers to enable more widespread use of serial crystallography methods for studies of metalloenzyme structure and protein dynamics. Strategies for time-resolved measurements and data analysis tools that provide rapid feedback for experimental optimization during fast-paced experiments will also be described.

Speaker: Aina Cohen (Stanford Synchrotron Radiation Lightsource)

15:10 Serial crystallography at the beamline P11, PETRA III

P11 at PETRA III (DESY, Hamburg) is a high-throughput instrument for macromolecular crys-
tallography [1]. The beamline has tuneable photon energy between 5.5 - 28 keV and and beam
sizes from 200 x 200 μm to 4 x 9 μm2 can be used with a maximum photon flux of 1x1013 ph/s at
12 keV energy. Equipped with a fast detector Eiger2 X 16M as the stationary detector (max 133 Hz),
this high flux instrument is perfect for serial synchrotron crystallography (SSX).
For SSX, the sample delivery is achieved through various types of solid supports and the TapeDrive
setup, which allows time-resolved room temperature experiments by the mix-and-diffuse method
[2, 3], and has been developed at P11 along with the real-time autoprocessing with CrystFEL [4,
5]. In this method, the samples are delivered on to a continuously drawn polyimide tape through
a 3D-printed microfluidic nozzle with two channels (ø 150 μm each), one for the crystal slurry and
second, for example, for mixing the crystals with a ligand or adjusting their pH. The mixing time
can be modified by the speed of the tape and the distance of the nozzle from the X-ray focus point
(delay times of 50 ms – 180 s).
In order to mitigate possible radiation damage, the exposure time per frame can be reduced from
7.5 ms instead of 3.5 ms with a chopper wheel. SSX experiments are currently controlled through
a separate graphical user interface, and online data analysis is available for real-time evaluation
and indexing via OnDA Monitor [6].
We will present the TapeDrive setup used for SSX, the real time data processing implemented as
well as several scientific examples exploiting the capabilities of the setup (time-resolved, mix-and-
diffuse experiments, temperature control…).
[1] Burkhardt, A., Pakendorf, T., Reime, B., Meyer, J., Fischer, P., Stübe, N., Panneerselvam, S.,
Lorbeer, O., Stachnik, K., Warmer, M., Rödig, P., Göries, D. & Meents, A. (2016). Eur. Phys. J. Plus
131, 56.
[2] Beyerlein, K. R., Dierksmeyer, D., Mariani, V., Kuhn, M., Sarrou, I., Ottaviano, A., Awel, S.,
Knoska, J., Fuglerud, S., Jönsson, O., Stern, S., Wiedorn, M. O., Yefanov, O., Adriano, L., Bean, R.,
Burkhardt, A., Fischer, P., Heymann, M., Horke, D. A., Jungnickel, K. E. J., Kovaleva, E., Lorbeer, O.,
Metz, M., Meyer, J., Morgan, A., Pande, K., Panneerselvam, S., Seuring, C., Tolstikova, A., Lieske,
J., Aplin, S., Roessle, M., White, T. A., Chapman, H. N., Meents, A. & Oberthuer, D. (2017). IUCrJ
4, 769.
[3] Henkel, A., Maracke, J., Munke, A., Galchenkova, M., Rahmani Mashhour, A., Reinke, P., Do-
maracky, M., Fleckenstein, H., Hakanpää, J., Meyer, J., Tolstikova, A., Carnis, J., Middendorf, P.,
Gelisio, L., Yefanov, O., Chapman, H. N. & Oberthür, D. (2022). Acta Cryst. A78, e560.
[4] White, T. A., Mariani, V., Brehm, W., Yefanov, O., Barty, A., Beyerlein, K. R., Chervinskii, F.,
Galli, L., Gati, C., Nakane, T., Tolstikova, A., Yamashita, K., Yoon, C. H., Diederichs, K. & Chapman,
H. N. (2016). J. Appl. Cryst. 49, 680.
[5] White, T. A. Schoof, T., Yakubov, S., Tolstikova, A., Middendorf, P., Karnevskiy, M., Mariani, V.,
Henkel, A., Klopprogge, B., Hannappel, J., Oberthür, D., De Gennaro Aquino, I., Egorov, D., Munke,
A., Sprenger, J., Pompidor, G., Taberman, H., Gruzinov, A., Meyer, J., Hakanpää, J., & Gasthuber,
G. (2025). IUCrJ 12, 97.
[6] Mariani, V., Morgan, A., Yoon, C. H., Lane, T. J., White, T. A., O’Grady, C., Kuhn, M., Aplin, S.,
Koglin, J., Barty, A. & Chapman, H. N. (2016). J. Appl. Cryst. 49, 1073.

Co-authors:
Alessandra Henkel, Julia Maracke, Spyridon Chatziefthymiou, Alexander Grebentsov, Andrey Gruzinov, Olga Merkulova, Philipp Middendorf, Alexandra Tolstikova, Thomas White, Dominik Oberthür, Johanna Hakanpää

Speaker: Guillaume Pompidor (DESY)

15:25 Time-resolved crystallography of light-driven ion transporters at synchrotrons and free electron lasers

Microbial rhodopsins constitute a large superfamily of light-sensitive membrane proteins, which are vital for numerous microorganisms on Earth, and for 20 years also being most-actively used in neuroscience and medicine in the biotechnology named optogenetics. The major role for optogenetics is played by ion-transporting rhodopsins. Understanding of their molecular mechanisms of functioning can not only contribute to the fundamental biological knowledge of their roles in native host, but can enable their optimization towards routine use in medical application, such as restoration of eye vision and hearing. I will report on our recent advances in investigations of light-driven ion pumps, with particular focus on applying time-resolved serial crystallography at X-ray free electron lasers and synchrotrons to obtain molecular movies of the proteins in action. These include tracking of ultrafast changes and slower but more prominent rearrangements in a new type of sodium transporters, as well as in inward proton pumps. I will also discuss the optimization of sample delivery for more efficient time-resolved crystallography experiments and its implication for other systems, such as near-infrared-absorbing microbial rhodopsins.

Speaker: Kirill Kovalev (EMBL-Hamburg)

15:55 Coffee break

16:30 Electrostatically Gated Enzyme Dynamics During Catalysis

Abstract: Enzyme catalysis is essential for life and is a central phenomenon in biochemistry. The advent of time-resolved serial crystallography, initially enabled by X-ray free electron lasers (XFELs) and now expanding to synchrotron X-ray sources, allows enzyme catalysis to be observed catalysis in real time, in near-physiological conditions, and at atomic resolution. I will describe our work using mix-and-inject serial crystallography (MISC) to observe catalysis by isocyanide hydratase (ICH). MISC allowed us to observe formation of an unusual thioimidate intermediate and to watch ICH’s conformational dynamics respond to changes in active site ionization during catalysis. We also used an engineered ICH mutant to enrich for rare conformations during catalysis, permitting a clearer view of later steps in the reaction. ICH exemplifies a class of enzymes whose non-equilibrium dynamics are gated by changes in active site electrostatics, which is a potentially common enzymological phenomenon.

Speaker: Mark Wilson (University of Nebraska–Lincoln)

17:00 Current and future capabilities for serial and time resolved crystallography at Diamond microfocus beamline VMXi

There is increasing interest in obtaining room temperature, time resolved crystal structures of
proteins carrying out their biological functions. The transition between conventional cryogenic
macromolecular crystallography and serial crystallography involving microcrystals remains chal-
lenging for many projects. We have recently demonstrated the capability to measure good quality,
low dose serial crystallography data from microcrystals within crystallisation plates [1]. This capa-
bility is available in the standard operation mode of the beamline and does not require any specific
serial crystallography apparatus. In this approach, microcrystals are transferred into a crystallisa-
tion plate (typically 100 nL per drop) and each droplet is subjected to raster scanning with a still
diffraction image measured every 10 µm. The resulting images are processed using standard serial
data processing software such as xia2.multiplex. This approach enables straightforward structure
determination and analysis of crystal quality and unit cell parameters from non-optimised crys-
tallisation conditions, guiding users in their optimisation efforts. Very small quantities of protein
are required, and the determination of a human peroxidase structure to 1.88 Angstrom resolution
using only 1.2 µL microcrystal suspension.
Several approaches to sample delivery for time resolved crystallography have been developed in-
cluding droplet-on-demand tape drive-based systems developed for XFEL experiments that have
been combined with X-ray emission spectroscopy (XES) to monitor the redox and spin state of
metal -containing cofactors within the proteins [2]. However, currently available systems require
a large quantity of microcrystal sample as well as requiring multiple skilled staff to operate. A
new system for serial crystallography at VMXi is currently under development. This incorporates
a picolitre droplet-on-demand tape drive system capable of anaerobic operation together with an
XES von Hamos spectrometer to enable spectroscopic validation in time resolved experiments of
metalloproteins. A compact design was required due to the tight spatial constraints of the VMXi
end station that was built for highly automated data collection from crystallisation plates, and the
design incorporates automation to reduce the number of personnel required to more closely ap-
proach a typical synchrotron experiment.
Proof of concept data obtained during the development process of the tape drive and XES spec-
trometer will be presented, including a high-resolution protein structure determined using the tape
drive system and XES data obtained from microcrystals of the copper enzyme nitrite reductase.
[1] A.J. Thompson, J. Sanchez-Weatherby, L.J. Williams, H. Mikolajek, J. Sandy, J.A.R. Worrall and
M.A. Hough (2024) Efficient in situ screening of and data collection from microcrystals in crystal-
lization plates Acta Cryst.D80, 279-288
[2] Butyrin, A. et al (2021) An on-demand, drop-on-drop method for studying enzyme catalysis by
serial crystallography. Nature Methods 12, 4461.

Authors:
Juan Sanchez Weatherby, Pierre Aller, Amy Thompson, Abby Telfer, John Sutter, James Sandy, Halina Mikolajek, Matthew Rodrigues, Mike Hough, Allen Orville

Speaker: Juan Sanchez Weatherby (Diamond Light Source)

17:15 → 19:50 Posters: Mingle & food

17:15 Posters & mingle food (Poster list with abstracts below)

17:30 In crystallo study of the reaction mechanism in a family B DNA polymerase

Reaction intermediates during DNA synthesis have been studied in detail using time-resolved
X-ray crystallography for translesion and repair DNA polymerases. Contrary to the originally
proposed two-metal-ion mechanism, a third metal ion was identified between the finger
domain and the α- and β-phosphates of the incoming nucleotide. This third metal ion was
suggested to either participate in catalysis or stabilize product formation. To investigate this
further in a replicative polymerase, we conducted time-resolved X-ray crystallography with
DNA Polymerase epsilon, which synthesizes DNA at a much faster rate—10x to 100x higher
than family Y and X polymerases. Surprisingly, no metal ion was observed between the finger
domain and the α- and β-phosphates of the incoming nucleotide in any of the solved structures
with Pol epsilon. Instead, our biochemical and structural data support the original two-metal
mechanism. In addition, we discovered that the 3’-OH group releases a proton, which is
channeled via structural waters to a basic residue in the Palm domain. After forming a new
bond with the incoming nucleotide, an acidic residue in the finger domain protonates the
released pyrophosphate, stabilizing the product. In summary, it seems that metal A’s role is
to lower the pKa of the 3’-OH group, followed by specific residues in Pol epsilon donating or
receiving a proton to catalyze this acid-base reaction.

Co-author: Erik Johansson

Speaker: Vimal Parkash (Umeå University)

17:40 Time-resolved serial crystallograhy to capture reaction intermediates of a glucuronyl esterase

Glucuronyl esterases (GEs) from the carbohydrate esterase family 15 (CE15) are involved in degrading lignocellulosic biomass, by catalyzing the hydrolysis 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 existing biological preteatment methods used in biorefineries to be able to make better use of this renewable energy source. The bacterial GE from Opitutus terrae (OtCE15A) has previously been structurally determined at cryo-temperature and a reaction mechanism for the acylation and deacylation reactions has been proposed (2,3). Various glucuronate- and galacturonate esters have been used as model substrates for the lignin-hemicellulose linkage, and the substrates have been soaked into the crystals. However, attempts to capture the binding of the substrates prior to hydrolysis of the ester bond have so far been unsuccessful, but a covalent reaction intermediate 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 initial time-resolved SSX experiment at P14.EH2 (T-REXX of PETRA III, Hamburg). We have obtained high resolution (1.7Å) SSX data of OtCE15A at BioMAX and observed binding of the cleaved substrate of benzyl glucuronoate after a soaking time of 5 minutes. For time-resolved SSX experiments at T-REXX, we have tested and are planning to use the hit-and-return (HARE) method (4).

References
1. Larsbrink, Johan, and Leila Lo Leggio. "Glucuronoyl esterases–enzymes to decouple lignin and carbohydrates and enable better utilization of renewable plant biomass." Essays in Biochemistry 67.3 (2023): 493-503.
2. Mazurkewich, Scott, et al. "Structural and biochemical studies of the glucuronoyl esterase OtCE15A illuminate its interaction with lignocellulosic components." Journal of Biological Chemistry 294.52 (2019): 19978-19987.
3. Zong, Zhiyou, et al. "Mechanism and biomass association of glucuronoyl esterase: an α/β hydrolase with potential in biomass conversion." Nature Communications 13.1 (2022): 1449.
4. Schulz, Eike C., et al. "The hit-and-return system enables efficient time-resolved serial synchrotron crystallography." Nature methods 15.11 (2018): 901-904.

Authors: Gabrielle Wehlander, Josefin Ridaeus, Scott Mazurkewich, Leila Lo Leggio, Johan Larsbrink, Gisela Brändén

Speaker: Gabrielle Wehlander (University of Gothenburg)

17:50 Comprehensive Support for Serial Crystallography at the European XFEL

Serial crystallography has opened new possibilities for determining the structures of biological macromolecules using microcrystals. At the European XFEL, the Sample Environment and Characterization (SEC) group, operating the XBI biology laboratory, provides essential support for users at every stage of the serial crystallography workflow, from initial sample preparation to advanced characterization and sample delivery.
The XBI laboratory offers state-of-the-art facilities where users can prepare and characterize their samples before experiments. Through hands-on support and guidance, we help users optimizing their samples to make efficient use of valuable beamtime. The SEC group provides sample delivery methods and supports users in optimizing their own systems.
By combining expertise in sample delivery and advanced characterization, the SEC group at European XFEL supports the structural biology community to achieve innovative results in serial crystallography. Our ongoing developments in standard samples, support infrastructure, and efficient sample delivery methods continue to lower access barriers, foster collaboration, and broaden the access to serial crystallography.

Speaker: Huijong Han (European XFEL)

18:00 Development of new data processing methods for serial time-resolved crystallography

To obtain the highest quality electron density maps, from a time-resolved serial crystallography experiment, it is crucial to accurately group the observed intensities according to the structure of the protein that generated those intensities. In an ideal world, each unique protein crystal structure would give rise to a set of well defined intensities, which would enable the intensities to be grouped easily. Unfortunately, due to many real world limitations, this is not the case: 1) The crystal is an average of many protein structures 2) Experimental effects such as unequal soaking, unequal laser/ x-ray exposure etc. 3) A range of crystal sizes causing inconsistent intensities which require rescaling. These effects give rise to uncertainty in the observed intensity values. Furthermore, only a minor proportion of hkls are affected by the change in protein structure, and of those hkls, the intensity changes are often subtle.
Despite 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 based approached used in many areas such as weather forecasting, econometrics and natural language processing. In principle, some Bayesian methods such 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 many nuances as to how this data processing pipeline should be set up, sometimes sacrificing flexibility for increased confidence in the results.

Co-authors: James Beilsten-Edmands, Mike Hough, Graeme Winter

Speaker: Rachel Tang (Diamond Light Source)

18:10 Structural studies of the human drug-metabolising protein CYP3A4

A highly flexible protein with an active site that changes its volume to fit a wide variety of ligands. A lid made of loops changes conformation based on ligand-size. Inhibition of this enzyme stops metabolism of drugs. This is an automatic disqualification of a drug candidate. Room temperature SSX shows better definition of some flexible loops, even at worse resolution. SSX data collection at tens of kilo Gray produces a similar active-site to our XFEL structure. I present an internal distance matrix analysis of a subset of PDB CYP3A4 structures to determine that crystal form, resolution and to some extent ligand-size dominates the clustering of global similarity with little difference caused by temperature. The protein crystalises as a monomer 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 initial data for the volume of the tetramer solved through single particle analysis at SciLifeLab Solna.

Co-authors: Owens Uwangue, Gisela Branden, Monika Bjelcic

Speaker: Johan Glerup (University of Gothenburg)

18:20 Scientific opportunities for Serial Crystallography at ALBA synchrotron

The ALBA Synchrotron, the 3 GeV light source in Southern Europe, is preparing for its upgrade to a 4th generation storage ring (ALBA II) together with the construction of 3 beamlines and major upgrades on the existing ones, early next decade. Within this framework, serial and time-resolved macromolecular crystallography (SSX and TR-MX) are identified as strategic growth areas.
BL13-XALOC, in operation since 2012 [1], has 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. Besides, the beamline is equipped with a high viscosity extrusion injector for SSX experiments. The transition from single crystal oscillation MX to SSX is highly simplified thanks a three-axis motorized stage and sample extrusion is facilitated thanks to a PID pressure control system. Proof-of-concept SSX experiments with test proteins have demonstrated compatibility with room-temperature data collection and negligible radiation damage [2]. Pump–probe TR-MX experiments using visible-light activation have also been successfully performed, establishing a baseline for further developments [3].
A second MX beamline, BL06-XAIRA, started regular user operation in June 2025, offering a highly stable microfocus beam of 3 × 1 μm² and photon energy range of <4.0–14 keV. Equipped with a <60nm runout diffractometer, an EIGER2 XE 9M and a dual Channel-Cut/multilayer monochromator [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 enable fixed-target SSX experiments at higher time resolution. Background reduction and long wavelength experiments are possible due to the recirculated He environment enclosing the entire end station.
The ALBA II upgrade plan foresees substantial investment in SSX/TR-MX capabilities for both beamlines. For XALOC, this includes hybrid tape-drive systems for tunable soaking times, and microfluidic injectors. XAIRA will focus on microfluidic chips, acoustic droplet ejection, and conveyor-belt systems for continuous fresh sample delivery. A pump-probe set up will be developed for both beamlines to allow for μs–ms TR-MX, including choppers, tunable lasers, and synchronization systems.
[1] Juanhuix, J., Gil-Ortiz, F., Cuní, G., Colldelram, C., Nicolas, J., Lidon, J., Boter, E., Ruget, C., Ferrer S. & Benach, J. (2014) Synchrotron Radiat. 21, 679-689.
[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., Grieco A., Ketawala G., Weierstall U., Spence J., Fromme P., Zatsepin N., Boer D.R. & Carpena X. (2022) J. Synchrotron Radiat. 29(3): 896-
[3] Kovalev K, et al, Nat Commun. 2020 May 1;11(1):2137.
[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:10.18429/JACoW-MEDSI2023-TUOAM04

Authors:
XAVI CARPENA I VILELLA, ISIDRO CRESPO
Co-authors:
DAMIÀ GARRIGA, FERNANDO GIL-ORTIZ, NAHIKARI GONZÁLEZ, ALEIX TARRÉS, ALBERT MIRET, BERNAT MOLAS, RICARDO VALCÁRCEL, ALEJANDRO ENRIQUE, CARLES COLLDELRAM, IGORS ŠICS, JOSÉ MARÍA ÁLVAREZ, MARCOS QUISPE, JULIÁN ALBERTO GARCÍA, ROELAND BOER, JUDITH JUANHUIX

Speakers: Isidro Crespo (ALBA-CELLS), Xavi Carpena i Vilella (ALBA-CELLS)

18:30 Reducing data volume with X-ray Laue diffraction

The X-ray Laue diffraction captures the crystal diffraction from white beam and can contain significant amount of structural information in comparison 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 data volume. Laue diffraction has great potential in mitigating the data volume 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 with 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 (iii) multi-crystal, ambient, single-frame approach with white beam. Compare to monochromatic diffraction, Laue diffraction dataset show increased content of structural information per image but with poor statistical metrics upon merging. Nevertheless, the solved structure from Laue diffraction dataset can resolve the duality of the loop conformation and reveal the loop conformational preference at productive state at ambient temperature. In short, Laue diffraction is a promising approach to explore in mitigating the data volume challenge.

Author: Kah Chee Pow
Co-author: Quan Hao

Speaker: Kah Chee Pow (Spallation Neutron Source Science Center)

18:40 Serial X – Simple Solutions for Serial Synchrotron Crystallography

Serial X aims to expand of the use of serial crystallography across the academic and industrial life-science community by introducing low cost, flexible and easy to use solutions to the problem of sample delivery in serial crystallography experiments at synchrotron radiation facilities. These solutions were invented in ProtonPump, the ERC Advanced Grant awarded to Richard Neutze, which applies 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 method has the potential to grow by up-to two orders of magnitude. This potential will never be realized if the current lack of standardization in sample delivery continues, which is prohibitively expensive and is often unreliable. Serial-X will solve this problem by developing and bringing to market standardized, low-cost, flexible and easy-to-use solutions for sample delivery in serial crystallography studies at synchrotron radiation sources. With the support of ERC proof-of-concept grant to Richad Neutze, Serial X has developed low-cost products supporting both flow cell1 and fixed target2 approaches to serial crystallography that are mounted upon standard magnets used at all conventional macromolecular X-ray crystallography synchrotron-based beamlines. This will remove the greatest obstacle currently preventing scientists from using serial crystallography for their own research or for structure based drug-design within a pharmaceutical drug discovery context.

Authors: Yanyan Chen, Swagatha Ghosh, Gisela Brändén, Richard Neutze

Speaker: Yanyan Chen (Serial X AB)

18:50 Experimental estimation of copper-ligand length precision in a model fungal LPMO under redox cycling and saccharide binding

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that degrade polysaccharides oxidatively, with applications in second-generation bioethanol production and as virulence factors in certain pathogens [1] [2]. They 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 chemically induced or triggered by photoreduction—can be subtle, the accurate determination of bond lengths and angles is essential [4]. In this study, LsAA9A from Lentinus similis was used as a model system under various experimental conditions. Analysis of the LsAA9A_Ec and LsAA9A_Ec_Cell3 structures collected under low-dose conditions showed that, compared with other Cu-coordination distances, only the Tyr–Cu bond exhibited a statistically significant change (p = 0.00094 in two tailed t-test). These findings confirm that saccharide substrate binding consistently shortens the Tyr–Cu distance in LsAA9A_Ec in the Cu²⁺ state, with a measured reduction of 0.21 Å. Furthermore, experiments on LsAA9A_Ec_Asc and LsAA9A_Ec_Asc_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 temperature, further probed structural responses to varying redox and experimental regimes. These findings advance the mechanistic understanding of LPMOs and offer a framework for probing subtle geometric changes in metalloenzymes.

Authors: Zhiyu Huang, Jie Nan, Monika Bjelcic, Leila Lo Leggio

Speaker: Zhiyu Huang (University of Copenhagen)

19:00 Time and dose resolved crystallography to control and capture redox states in heme peroxidases

Metalloenzymes containing heme centres catalyse a wide range of reactions critical to life. Understanding the structure and electronic states of the heme centre across multiple functionally relevant states is essential to understand mechanism. 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, complementary, serial crystallography and single-crystal spectroscopic approaches.

Fixed target drop-on-chip, tape drive droplet on demand and correlated spectroscopies allow the formation of high valent Fe(IV) states to be characterised. X-ray Pump Probe serial femtosecond crystallography (SFX) together 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 response of the Fe(IV)-O state is highly variable between the chemically identical heme groups of the homo-oligomeric proteins highlighting the importance of understanding the effect of the crystalline lattice on observed changes in time- and dose-resolved crystallography experiments.

Lucic, M. et al (2021) Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases. JBIC 27 (7), 743-761.

Co-authors: Robin Owen, Jonathan Worrall, Marina Rozman, Lewis Williams, Danny Axford, Allen Orville, Pierre Aller, Jan Kern, Jos Kamps

Speaker: Michael Hough (Diamond Light Source)

19:10 Standard Sample Preparation and Characterization for Serial Crystallography

Serial crystallography (SX) has been a revolutionary technique in structural biology for more 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 possible, avoiding radiation damage and allowing for the capture of transient states and intermediates in biological reactions. The success of SX experiments heavily relies on the quality of the sample.
Standard samples in SX research provide critical roles. First, the commissioning of new beamline devices needs well-characterized samples. By providing consistent and reproducible diffraction patterns, the standard samples help in the calibration of new devices and verification of their performance. Secondly, they are required for the validation of experimental setups, ensuring that all components, from sample delivery systems to data acquisition software, function correctly.
In this study, we present detailed protocols for the preparation of lysozyme, myoglobin, iq-mEmerald, and photoactive yellow protein (PYP) crystals. These proteins were selected as standard samples due to their robust crystallization properties and suitability for a wide range of SX experiments. Through the optimization of existing protocols, we achieved high-quality crystal samples with improved yield, specifically for SX applications.

Authors: Christina Schmidt, Huijong Han

Speaker: Christina Schmidt (European XFEL)

19:20 Laser and Spectroscopic Capabilities at MicroMAX

Spectroscopic techniques provide a powerful means of obtaining detailed information on the structural and dynamic properties of proteins in solution and in crystallo (proteins and enzymes are active in the crystalline state). Structural data obtained by X-ray crystallography is strengthened by insights from complementary approaches like UV/vis, fluorescence and (resonance) Raman measurements.

Speakers: Manoop Chenchiliyan (MAX IV Laboratory), Sofia M. Kapetanaki (MAX IV Laboratory)

19:30 MicroMAX - A beamline with time-resolved macromolecular crystallography capabilities at the MAX IV Laboratory

The rise of 4th generation sources, including the MAX IV Laboratory 3 GeV ring, has enabled new possibilities to study dynamics using crystallography. The MicroMAX beamline is a new beamline focussed on providing optimal X-ray characteristics for serial (SSX) and time-resolved (TR-SSX) crystallography at MAX IV [1]. The beamline emphasizes a flexible sample environment for standard and bespoke experimental setups while also supporting high-throughput single crystal data collections at the BioMAX beamline which has operated since 2017 [2].
The MicroMAX user program opened in May 2024 and has performed experiments with SPINE-based fixed targets, high-viscosity extrusion and microfluidics and single-crystal oscillation data collections. Sample handling and positioning is supported by the MD3-up micro diffractometer, Oxford cryojet, and ISARA automated sample mounting platform (including crystallization plates). Time resolved techniques are enabled by a nanosecond pump laser (210-2600 nm), Celerotron X-ray chopper (0,8-70% duty cycle) and one of either an Eiger2 X 9M CdTe photon counting hybrid pixel detector or Jungfrau 9M Si integrating hybrid pixel detector (on-loan from PSI).
Optical 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 using beryllium lenses/K-B mirrors. Beamline controls are from within MXCuBE, with additional live feedback and CrystFEL autoprocessing pipelines to provide immediate feedback and rapid map generation. Sample pre-characterization is supported by an offline laser and spectroscopy lab in the secondary experimental hutch and dedicated sample environment and preparation labs.
Here we present the current status of MicroMAX beamline and recent developments in sample preparation and data handling under a variety of experimental contexts. This work emphasizes the technical developments for a highly flexible TR-SSX end station in context of SSX/TR-SSX experiments already being conducted by the MicroMAX user community.

MicroMAX is funded by the Novo Nordisk Foundation under the grant number NNF17CC0030666.

[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., Aurelius, O., Lang, D., Ursby, T. & Thunnissen, M. M. G. M. (2025). J. Synchrotron Rad. 32.
[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.

Speaker: Jie Nan et al (MAX IV Laboratory)

19:40 Fragment Based Active Site Exploration of Polyurethane Degrading Enzymes for Structure-guided Protein Engineering

Polyurethane (PU) plastics are extermyl durable and hard to recycle by convential methods due to
crosslinked and branched molecular structure. Recent discoveries of enzyme that arget carbamate
(urethane) bond in the PU provide alternative way of PU recycling by means of biocatalytic degradation.
EnZync center has been established to discover, characterize and engineer novel enzymes for PU
degradation. So far we have discovered several enzymes by computational searches and experimental
ways. The critical steps to establish rational basis of protein engineering campaing is the understanding
of Plastic-enzyme interactions at molecular level. To pursue structural characterization of “PURases”
we have two different but complemantry methods to characteirze active site of PURases: i) Fragment
Based Active site Exploration (FASE) of PURases by using small molecule fragment libraries and
soluble PU analogs and ii) time-resolved serial crystallography by cryocapturing catalytic intermideate
(for slow-milisecond kinetics) and ambient temperature (fast-microsecond) crystallography. We have
completed FASE approaches for one of our patented enzymes and now we are strating to perform serial
crystallography approach with promising preliminary data.

Authors: Deniz Bicer, Laura Rotilio, Daniel Otzen & Jens Preben Morth

Speaker: Deniz Bicer (Aarhus University)

Tuesday 23 September

08:30 → 14:30 Tuesday: Morning & early afternoon

08:30 Arrival and coffee

09:00 Time-resolved X-ray crystallography on membrane proteins: Watching ions moving in time and space

P. Nogly
Dioscuri Centre for Structural Dynamics of Receptors, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; przemyslaw.nogly@uj.edu.pl

Chloride transport is an essential process maintaining ion balance across cell membranes, cell growth, and neuronal action potentials. However, the molecular mechanism of the transport remains elusive. Among chloride transporters, light-driven rhodopsins have gained attention as optogenetic tools to manipulate neuronal signaling. We combined time-resolved serial crystallography at XFEL and synchrotron to provide a comprehensive view of chloride-pumping rhodopsin's structural dynamics and molecular mechanism throughout the transport cycle from 10 ps to 50 ms [1]. We traced transient anion binding sites, obtained evidence for the mechanism of light energy utilization in transport, and identified steric and electrostatic molecular gates ensuring unidirectional transport. These structural insights provided the basis for mutagenesis and functional study of the mechanistic features enabling finely controlled chloride transport across the cell membrane.
Furthermore, our recent study of a distinct photoreceptor, Light-Oxygen-Voltage (LOV) domain, will be introduced. The first insights into the structural dynamics of LOV photoactivation will be presented, providing the basis for proposing a molecular mechanism of a covalent thioether bond formation between a flavin mononucleotide cofactor and a reactive cysteine, Cys57 (unpublished).

[1] Mous, S. et al. Science 375 (2022) 845

Speaker: Przemyslaw Nogly (Jagiellonian University in Krakow)

09:30 Using advanced crystallographic approaches to resolve how orange carotenoid protein photocycle works

Speaker: Volha Chukhutsina (Vrije Universiteit Amsterdam)

10:00 GPCRs as Targets for Serial Crystallography

Using time-resolved serial crystallography to observe structural snapshots of protein dynamics at high resolution is a method that is becoming gradually more commonplace. Advancements in method development for this technique have allowed a wider range of proteins to be studied; looking at processes spanning endogenous photoresponses, enzyme kinetics and ligand binding. G protein-coupled receptors represent a pharmacologically relevant superfamily of proteins that are interesting targets for study with time-resolved serial crystallography. Data from time-resolved serial crystallography has the potential to enhance the drug design process by revealing protein transitional states that can be either targeted or used to provide information about protein flexibility. Our goal is to study the inherent dynamics of GPCRs critical for receptor function and to use this information to develop more targeted ligands. Here, we present the results of time-resolved serial crystallography experiments conducted at MaxIV and the SLS on the human A2a receptor. Through synthetic photoswitches, based on the marketed drug istradefylline for the treatment of Parkinson’s disease, light is used as a trigger to investigate the dynamics associated with ligand dissociation from the receptor orthosteric binding pocket. Our time-resolved data highlights key structural features involved in the transition upon ligand photoswitching. This includes the rearrangement of extracellular loops 2 and 3 that form a lid over the binding pocket, which has been shown by molecular dynamic simulations, crystal structures and kinetic analyses to be crucial for ligand dissociation and long target resident time. Additionally, lessons learned from this investigation, in terms of experimental design and sample preparation, can be applied to future projects using GPCRs as targets for serial crystallography. Helping to lower the barrier of entry to time-resolved serial crystallography and ultimately leading to more rationally designed drugs.

Co-authors:
Torben Saßmannshausen, Quentin Bertrand, Matilde Trabuco, Chavdar Slavov, Arianna Bacchin, Fabio Andres, Yasushi Kondo, Robin Stipp, Maximilian Wranik, Georgii Khusainov, Melissa Carrillo, Demet Kekilli, Jie Nan, Ana Gonzalez, Robert Cheng, Werner Neidhart, Tobias Weinert, Filip Leonarski, Florian Dworkowski, Michal Kepa, Josef Wachtveitl, Michael Hennig, Joerg Standfuss

Speaker: Hannah Glover (leadXpro)

10:15 Coffee break

10:45 Serial femtosecond crystallography of high-valent metal sites and protein radicals

Högbom, M 1

1. Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden.

High resolution structure determination methods suffer from problems with radiation damage. This is particularly problematic for radiation sensitive states such as high-valent metal sites and radicals. From a chemical perspective this means that some of the most relevant states for catalysis in many enzyme systems are inaccessible to standard structure determination regimes.
In close collaboration with scientists at the LCLS and the LBNL we utilize a conveyor-belt sample injector that allows micrometer-sized crystals to be manipulated in various ways, including oxygen incubation for a defined period of time, before exposure to the free-electron laser X-ray beam [1]. This setup allows varying the time for intermediate trapping while the use of femtosecond XFEL crystallography eliminates the effect of X-ray photoreduction on obtained data. Simultaneous XES also allows in situ oxidation state determination of probed intermediates for metalloprotein systems.
This setup and its use to obtain high-resolution global geometric structures of high-valent intermediates will be discussed, as well as our recent progress defining radiation undamaged structures of methane monooxygenase [2] and ribonucleotide reductase R2 proteins [3,4] including the catalytic radical state [5].

Acknowledgments: This work was funded by the Knut and Alice Wallenberg Foundation, the Swedish Research Council and the European Research Council (ERC).

References
[1] F.D. Fuller et al. Nature Methods, 14(4):443-449 (2017)
[2] Srinivas V. et al. J Am Chem Soc, 142:14249-14266 (2020)
[3] Srinivas V. et al. Nature, 563:416-420 (2018)
[4] John J. et al. Elife, 11:e79226 (2022)
[5] Lebrette H. et al. Science, 382:109-113 (2023)

Speaker: Martin Högbom (Stockholm University)

11:15 Modeling X-ray-Induced Heating at 4th-Generation MX Beamlines

The transition from 3rd to 4th generation synchrotrons—featuring diffraction-limited storage rings—has significantly expanded future possibilities for macromolecular crystallography (MX). These upgrades, characterized by reduced source divergence and increased electron bucket capacity, have boosted brilliance by up to two orders of magnitude. As a result, next-generation MX beamlines, such as ID29 (ESRF-EBS), BioCARS (APS-U), and MicroMAX (MAX IV), now deliver fluxes approaching 10^15 ph/s. These extremely high flux (EHF) beamlines are increasingly optimized for time-resolved MX, aiming for microsecond-scale resolution.

Operating in previously unexplored dose regimes (>50 GGy/s) raises new challenges. In my presentation, I would like to share our study that focuses on beam-induced heating in microcrystals (<25 µm) exposed to EHF conditions. Thermal modeling indicates that such dose rates may cause significant temperature rises, potentially impacting data quality. Mitigation strategies include using top-hat beam profiles and increasing both beam and crystal sizes to distribute dose more evenly. The proposed model serves as a tool to support experimental design and optimize conditions for high-flux time-resolved MX. This of critical importance for future multidimensional X-ray protein crystallography, especially in enzymology, where temperature is a key experimental variable. As such they will require precise temperature measurements, as even a small change in temperature can affect the catalytic activity of an enzyme.

Authors: Michal Kepa, John Beale, Martin Appleby

Speaker: Michal Kepa (Paul Scherrer Institute)

11:45 Quantum refinement used for time-resolved crystallography

In standard crystallographic refinement of proteins, the experimental data are normally not enough to unambiguously decide the positions of all atoms. Therefore, the crystallographic data are supplemented by a set of empirical restraints that ensure that bond lengths and angles make chemical sense. To obtain more accurate results, we have suggested that this potential can be replaced by more accurate quantum-mechanical (QM) calculations for a small, but interesting part of the protein, giving the method of quantum refinement.1 Our group has shown that quantum refinement can locally improve crystal structures,2 decide protonation state of metal-bound ligands,3–6 oxidation state of metal sites,7,8 detect photoreduction of metal ions7,9 and solve scientific problems regarding what is really is seen in crystal structures.9–11 Several other groups have implemented this and similar approaches.12 We investigate how quantum refinement can be used for time-resolved crystallography. In time-resolved crystallography, the obtained electron-density maps will typically involve a mixture of several states (unreacted state, intermediates and products). Therefore, the structures will heavily depend on the empirical potential and the expectations of the crystallographer. The QM calculations will give more accurate results, especially if there are intermediates with unusual (e.g. twisted) structures or if metal sites are involved (which are hard to describe with general restraints). Moreover, we will couple the structural interpretations with expectations from kinetic models of the studied reaction. I will present some preliminary applications on cytochrome c oxidase, xylose isomerase and bacteriorhodopsin.

References
1. U. Ryde, L. Olsen, K. Nilsson, 2002, J. Comput. Chem. 23, 1058.
2. U. Ryde, K. Nilsson J. Am. Chem. Soc. 2003, 125, 14232.
3. K. Nilsson, U. Ryde, J. Inorg. Biochem., 2004, 98, 1539
4. L. Cao, O. Caldararu, U. Ryde, J. Phys. Chem B, 2017, 121, 8242.
5. L. Cao, O. Caldararu, U. Ryde, J. Chem. Theory Comput., 2018, 14, 6653.
6. O. Caldararu, M. Feldt, D. Cioloboc, M.-C.van Severen, K. Starke, E. Nordlander, et al. Sci. Rep. 2018, 8, 4684
7. L. Rulíšek, U. Ryde, J. Phys. Chem. B, 2006, 110, 11511
8. L. Cao, Börner, M. C., Bergmann, J., Caldararu, O. & U. Ryde, Inorg. Chem. 2019, 58, 9672.
9. P. Söderhjelm, U. Ryde, J. Mol. Struct. Theochem, 2006, 770, 199
10. L. Cao, O. Caldararu, A. C. Rosenzweig, U. Ryde, 2018, Angew. Chem. Int. Ed., 57,162.
11. J. Bergmann, E. Oksanen & U. Ryde, J. Biol. Inorg. Chem. 2021, 26, 341.
12. J. Bergmann, E. Oksanen, U. Ryde, Curr. Opin. Struct. Biol. 2022, 72, 18.

Authors:
Gayathri Yuvaraj, Ulf Ryde
Co-authors:
Kristoffer Lundgren, Esko Oksanen

Speaker: Gayathri Yuvaraj (Lund University)

12:00 Lunch and poster session

13:30 MicroMAX - Overview

The rise of 4th generation sources, including the MAX IV Laboratory 3 GeV ring, has enabled new possibilities to study dynamics using crystallography. The MicroMAX beamline is a new beamline focussed on providing optimal X-ray characteristics for serial (SSX) and time-resolved (TR-SSX) crystallography at MAX IV [1]. The beamline emphasizes a flexible sample environment for standard and bespoke experimental setups while also supporting high-throughput single crystal data collections at the BioMAX beamline which has operated since 2017 [2].
The MicroMAX user program opened in May 2024 and has performed experiments with SPINE-based fixed targets, high-viscosity extrusion and microfluidics and single-crystal oscillation data collections. Sample handling and positioning is supported by the MD3-up micro diffractometer, Oxford cryojet, and ISARA automated sample mounting platform (including crystallization plates). Time resolved techniques are enabled by a nanosecond pump laser (210-2600 nm), Celerotron X-ray chopper (0,8-70% duty cycle) and one of either an Eiger2 X 9M CdTe photon counting hybrid pixel detector or Jungfrau 9M Si integrating hybrid pixel detector (on-loan from PSI).
Optical 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 using beryllium lenses/K-B mirrors. Beamline controls are from within MXCuBE, with additional live feedback and CrystFEL autoprocessing pipelines to provide immediate feedback and rapid map generation. Sample pre-characterization is supported by an offline laser and spectroscopy lab in the secondary experimental hutch and dedicated sample environment and preparation labs.
Here we present the current status of MicroMAX beamline and recent developments in sample preparation and data handling under a variety of experimental contexts. This work emphasizes the technical developments for a highly flexible TR-SSX end station in context of SSX/TR-SSX experiments already being conducted by the MicroMAX user community.

MicroMAX is funded by the Novo Nordisk Foundation under the grant number NNF17CC0030666.

[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., Aurelius, O., Lang, D., Ursby, T. & Thunnissen, M. M. G. M. (2025). J. Synchrotron Rad. 32.
[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.

Speaker: Jie Nan (MAX IV Laboratory)

13:50 Sample delivery at MicroMAX

The rise of 4th generation sources, including the MAX IV Laboratory 3 GeV ring, has enabled new possibilities to study dynamics using crystallography. The MicroMAX beamline is a new beamline focussed on providing optimal X-ray characteristics for serial (SSX) and time-resolved (TR-SSX) crystallography at MAX IV [1]. The beamline emphasizes a flexible sample environment for standard and bespoke experimental setups while also supporting high-throughput single crystal data collections at the BioMAX beamline which has operated since 2017 [2].
The MicroMAX user program opened in May 2024 and has performed experiments with SPINE-based fixed targets, high-viscosity extrusion and microfluidics and single-crystal oscillation data collections. Sample handling and positioning is supported by the MD3-up micro diffractometer, Oxford cryojet, and ISARA automated sample mounting platform (including crystallization plates). Time resolved techniques are enabled by a nanosecond pump laser (210-2600 nm), Celerotron X-ray chopper (0,8-70% duty cycle) and one of either an Eiger2 X 9M CdTe photon counting hybrid pixel detector or Jungfrau 9M Si integrating hybrid pixel detector (on-loan from PSI).
Optical 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 using beryllium lenses/K-B mirrors. Beamline controls are from within MXCuBE, with additional live feedback and CrystFEL autoprocessing pipelines to provide immediate feedback and rapid map generation. Sample pre-characterization is supported by an offline laser and spectroscopy lab in the secondary experimental hutch and dedicated sample environment and preparation labs.
Here we present the current status of MicroMAX beamline and recent developments in sample preparation and data handling under a variety of experimental contexts. This work emphasizes the technical developments for a highly flexible TR-SSX end station in context of SSX/TR-SSX experiments already being conducted by the MicroMAX user community.

MicroMAX is funded by the Novo Nordisk Foundation under the grant number NNF17CC0030666.

[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., Aurelius, O., Lang, D., Ursby, T. & Thunnissen, M. M. G. M. (2025). J. Synchrotron Rad. 32.
[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.

Speaker: Dean Lang (MAX IV Laboratory)

14:10 Data analysis at MicroMAX

The rise of 4th generation sources, including the MAX IV Laboratory 3 GeV ring, has enabled new possibilities to study dynamics using crystallography. The MicroMAX beamline is a new beamline focussed on providing optimal X-ray characteristics for serial (SSX) and time-resolved (TR-SSX) crystallography at MAX IV [1]. The beamline emphasizes a flexible sample environment for standard and bespoke experimental setups while also supporting high-throughput single crystal data collections at the BioMAX beamline which has operated since 2017 [2].
The MicroMAX user program opened in May 2024 and has performed experiments with SPINE-based fixed targets, high-viscosity extrusion and microfluidics and single-crystal oscillation data collections. Sample handling and positioning is supported by the MD3-up micro diffractometer, Oxford cryojet, and ISARA automated sample mounting platform (including crystallization plates). Time resolved techniques are enabled by a nanosecond pump laser (210-2600 nm), Celerotron X-ray chopper (0,8-70% duty cycle) and one of either an Eiger2 X 9M CdTe photon counting hybrid pixel detector or Jungfrau 9M Si integrating hybrid pixel detector (on-loan from PSI).
Optical 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 using beryllium lenses/K-B mirrors. Beamline controls are from within MXCuBE, with additional live feedback and CrystFEL autoprocessing pipelines to provide immediate feedback and rapid map generation. Sample pre-characterization is supported by an offline laser and spectroscopy lab in the secondary experimental hutch and dedicated sample environment and preparation labs.
Here we present the current status of MicroMAX beamline and recent developments in sample preparation and data handling under a variety of experimental contexts. This work emphasizes the technical developments for a highly flexible TR-SSX end station in context of SSX/TR-SSX experiments already being conducted by the MicroMAX user community.

MicroMAX is funded by the Novo Nordisk Foundation under the grant number NNF17CC0030666.

[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., Aurelius, O., Lang, D., Ursby, T. & Thunnissen, M. M. G. M. (2025). J. Synchrotron Rad. 32.
[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.

Speaker: Cecilia Casadei (MAX IV Laboratory)

14:30 → 17:00 Tuesday afternoon

14:30 Panel discussion

Moderator: Dr. John Beale, Paul Scherrer Institute

Prof. Guillermo Montoya, University of Copenhagen
Prof. Gisela Brändén, University of Gothenburg
Dr. Judith Juanhuix, ALBA-CELLS

Speaker: John Beale (Paul Scherrer Institute)

15:00 Coffee and move to MAX IV

15:45 MAX IV visit

19:00 → 22:00 Tuesday afternoon: Symposium Dinner

Wednesday 24 September

08:30 → 13:00 Morning: Wednesday

08:30 Arrival and coffee

09:00 Investigating enzyme mechanisms by multidimensional crystallography

Functional characterization of proteins requires linking structure and dynamics, but traditional X-ray crystallography provides only static snapshots. Serial time-resolved crystallography enables direct visualization of structural changes over time, including internal motions and solvent interactions. We developed fixed-target approaches such as “Hit And REturn” (HARE) and reaction initiation strategies using piezo droplet injectors. The “Liquid Application Method for time-resolved Analysis” (LAMA) further broadens applicability to systems not triggered by light. In addition, environmental control allows temperature variation from 7 °C to 70 °C, enabling multi-dimensional experiments. These advances permit direct observation of ligand binding, intermediates, and conformational changes, as demonstrated by tracking glucose-to-fructose conversion in Xylose isomerase across both temperature and time. Together, these methods expand the toolkit for time-resolved crystallography, opening the way to mechanistic insights into enzyme dynamics, allostery, and solvent networks.

Speaker: Pedram Mehrabi (University of Hamburg)

09:30 Time-resolved macromolecular crystallography studies of AmpCEC using synchrotron and XFEL radiation

The β-lactamase enzymes degrade β-lactam antibiotics, exemplified by penicillin. As such, the families of metallo- and serine β-lactamases are responsible for a major antimicrobial resistance mechanism in many clinically relevant species of Gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. To preserve the antimicrobial activity of β-lactam antibiotics, inhibitors of β-lactamases can be used in combination with a β-lactam antibiotic during treatment of an antimicrobial resistant infection. However, these inhibitors often have a narrow spectrum of activity against β-lactamases, and other bacterial mechanisms of resistance against them. To further development of novel β-lactamase inhibitors, we apply time-resolved serial femtosecond and synchrotron crystallography (tr-SF/SX) techniques to investigate the acylation of the β-lactamase AmpC from Escherichia coli by avibactam, a clinically approved β-lactamase inhibitor. Previously, tr-SF/SX data gathered by using a “drop-on-demand” sample delivery system revealed that covalent binding of avibactam to the AmpC active site occurred in a time frame quicker than 200 ms and as such pre-acylated time-resolved structures could not be obtained using this system. In the interest of capturing earlier time points using this enzymatic system, we have turned our focus to using a “drop-on-chip” fixed target sample delivery system, addressing contamination issues and implementing robust controls in our setup for accurate data collection. Currently, we are employing a drop-on-chip fixed target sample delivery system to access time points >1 ms at XFELs and >10 ms at synchrotrons such as Diamond Light Source.

Authors:
Emily Freeman, Jos Kamps, Pierre Aller, Christopher Schofield, Allen Orville

Speaker: Emily Freeman (University of Oxford)

09:45 Coffee break

10:15 Turning Up the Heat on Dynamic Proteins with Temperature-Jump X-ray Crystallography

Protein dynamics are critical for function, but it remains challenging to understand, in atomic detail, how a molecule’s biological activity is enabled by the physical coupling of its conformational fluctuations across varied length and time scales. Time-dependent X-ray crystallographic measurements of molecular structure can overcome some of the limitations of traditional structural biology and yield deep insight into protein conformational landscapes, but it remains challenging to initiate synchronous conformational changes in crystallized macromolecules, which is a requirement for such experiments. I will describe how observations from multi-temperature structural measurements motivated the development of temperature-jump (T-jump) crystallography, and summarize the results of our early T-jump experiments on the model enzyme lysozyme. I will also discuss ongoing efforts to democratize these experiments and apply them to increasingly complex biological systems, including the metalloenzyme soybean lipoxygenase, whose catalytic mechanism involves a rate-limiting hydrogen tunneling step that is coupled to motion of the protein scaffold.

Speaker: Michael Thompson (University of California, Merced)

10:45 Serial Techniques for Weakly Scattering, In-Situ, and Dynamic Systems

Speaker: Mark Warren (Diamond Light Source)

11:15 Serial Microsecond Crystallography with the ESRF Extremely Brilliant Source

Time-resolved serial crystallography (TR-SX) is a leading technique for capturing biological processes as molecular movies on extremely fast timescales—fulfilling a long-standing goal in structural biology. The method involves delivering microcrystals into a powerful, pulsed X-ray beam to collect individual diffraction patterns from each crystal. By compiling thousands of these patterns, researchers can reconstruct an electron density map. This technique, known as serial crystallography, allows for the observation of structural changes. When ultrashort X-ray pulses are used, SX can track time-dependent conformational changes, enabling scientists to visualize proteins in motion. TR-SX experiments are typically synchronized with a specific stimulus that initiates the biological activity under investigation. The advent of diffraction limited storage rings - the so-called 4th generation synchrotrons - have permitted the conception and built instruments that overcomes the limits of traditional microfocus beamlines. These new beamlines aim to exploit X-ray pulses down to microsecond time resolution, becoming an invaluable tool for room temperature and time-resolved studies that complements the capabilities of Free Electron Laser sources. ID29 at the European Synchrotron Radiation Facility is one of the first examples of this new generation of beamlines (Orlans et al. 2025). The unique combination of microsecond exposure times, advanced beam properties, and a flexible sample environment enables the collection of high-quality, complete data—even from exceptionally small amounts of crystalline material. This is applied in combination with external stimuli to activate or induce structural changes that could be observed in the microsecond time regime. This approach is particularly successful for the study of enzymatic reaction or ligand binding, thus prominently interesting for the whole structural biology community, while future developments will be crucial to strengthen the application of the methods to structural based drug design.

Orlans, J. (2025). Advancing macromolecular structure determination with microsecond X-ray pulses at a 4th generation synchrotron. Communications Chemistry, 8(1), 1–12. https://doi.org/10.1038/s42004-024-01404-y

Speaker: Daniele de Sanctis (ESRF - The European Synchrotron)

11:45 Wrap-up

12:00 Lunch

13:00 → 14:00 End of Symposium: Start of TR+ Workshop