Illuminating Biomolecular Complexity: X-ray Free Electron Lasers and Vibrational Spectroscopies for Protein, Aggregates, and Cellular Architectures

Jun 29, 2025, 8:00 AM → Jun 30, 2025, 10:15 PM Europe/Rome

Angela Capocefalo, Ines Delfino, Emiliano De Santis (Istituto Nazionale di Fisica Nucleare), Valentina Notarstefano, Francesco Stellato (Istituto Nazionale di Fisica Nucleare)

Satellite meeting to the 15th European Biophysical Congress  

X-ray Free Electron Lasers (XFELs) provide ultra-bright photon pulses to perform diffraction, scattering and spectroscopy measurements on a wealth of biological systems, going from small proteins up to organelles and cells. In particular, vibrational spectroscopies exploit photons coming from different sources to probe chemical bond vibrations, yielding static and time-resolved pictures of biological samples.

Key topics of this workshop include time-resolved XFEL experiments and vibrational spectroscopy measurements, including recent ultra-fast developments, to study protein dynamics, biomolecular assemblies and disease-related aggregates. Emphasis will be placed on discussing synergistic approaches between XFEL measurements and vibrational spectroscopies, taking into account the most recent advancements and their applications in structural biology and biophysics.

This satellite meeting to the EBSA congress aims to foster collaborations among experimentalists, computational, and photon sources developers who are already part of or closely connected to the EBSA community, while also encouraging those who are not yet members to join. By bridging the diverse fields, the workshop aims at opening new frontiers in biophysical research, combining diverse expertise to tackle complex biological questions with cutting-edge technologies.

Sunday, June 29

9:30 AM → 12:55 PM Session 1

9:40 AM Welcome

9:45 AM Orienting gas-phase proteins with electric fields for X-ray imaging

Using X-ray diffraction for structure determination is further complicated when the irradiated particles have random and unknown orientations, because it means that the relations between the diffraction images are also unknown. Algorithms exist for recovering the relative orientations between diffraction patterns, but they do not always converge, especially not when faced with scarce or noisy data. Controlling the orientation of the particle would help overcome this problem in single particle imaging and related techniques. We once demonstrated the possibility of orienting proteins in the gas phase without destroying their structures using strong electric fields via the interaction with their electric dipole moments. More recently we have explored more aspects of dipole orientation, including if and how it actually helps the orientation recovery, and how the orientation is affected by a thin layer of water around the protein, which has been shown to have other benefits for single particle imaging. We hope that our results can serve to guide the development of new technology and experiments that utilise dipole orientation for structure determination of macromolecules. To that end, we here present our current research in dipole orientation of gas-phase proteins.

Speaker: Erik Marklund (Uppsala University)

10:20 AM MS SPIDOC: Coherent diffractive imaging of proteins and viral capsids

MS SPIDOC is an innovative sample delivery system tailored for single-particle imaging at X-ray Free-Electron Lasers (XFELs) and adaptable to most large-scale facility beamlines. It accommodates a wide range of biological samples, from small proteins to megadalton (MDa) complexes. Utilizing nano-electrospray ionization, ionic samples are $m/z$-filtered and structurally separated before reaching the interaction zone for imaging. We present the first proof-of-principle experiments demonstrating gas-phase small-angle X-ray scattering (SAXS) at the PETRA III synchrotron (DESY, Germany), highlighting the system’s potential for advancing structural biology in the gas phase.

Speaker: Thomas Kierspel

10:40 AM Investigating the Origins of Non-Aromatic Fluorescence with Vibrational and X-ray Absorption Spectroscopies

Non-aromatic fluorescence in biomolecules represents a fascinating photophysical phenomenon that challenges conventional understanding of fluorescent mechanisms.. This presentation outlines our ongoing investigation using a combined approach of time-resolved X-ray absorption spectroscopy (TrXAS) and multiple vibrational spectroscopic techniques to explore the fundamental processes governing this phenomenon.

Our research examines L-glutamine (L-glu) and its derivative L-pyroglutamine ammonium (L-pyro-amm), where we hypothesize that hydrogen bonding networks may influence conical intersections (CoIns) and thus modulate nonradiative decay pathways. The complementary techniques of FTIR, far-IR, and Raman spectroscopy are being employed to characterize structural dynamics, while TrXAS measurements at the carbon K-edge at the EIS-TIMEX FEL beamline are anticipated to provide insights into excited-state evolution.

This presentation will discuss our methodological approach, preliminary observations, and the theoretical framework guiding our investigation. We will consider how this integrated spectroscopic strategy may contribute to elucidating the structural and electronic factors that enable non-aromatic fluorescence, with potential implications for the future development of novel fluorescent biomaterials and optical probes.

Speaker: Zeinab Ebrahimpour (Elettra-Sincrotrone Trieste, Area Science Park Basovizza, 34149, Trieste, Italy & INFN)

11:25 AM Vibrational Spectroscopy to Tackle Cancer

Normal-to-cancer transition (NTC) is still an ill-understood process, closely associated to cellular biomechanical properties. These are strongly dependent on intracellular water´s structural and dynamical profiles, which play a fundamental role in cellular function. Improved chemotherapeutic strategies are an urgent clinical need, since cancer is still the second leading cause of death worldwide, with an expected rising incidence. Metal-based drugs developed upon the discovery of cisplatin (cis-(NH3)2PtCl2) have aimed at coupling an enhanced efficacy to decreased acquired resistance and harmful side effects. These metallodrugs encompass Pt- and Pd-complexes with more than one metal centre [1], extensively studied by the authors in the last decade [2-9], which trigger a selective DNA damage – through metal coordination to the purine bases or via electrostatic interaction with the phosphate groups.
Inelastic and quasi-elastic neutron scattering techniques (INS and QENS), combined with Raman and Fourier Transform Infrared (FTIR, including with synchrotron radiation) spectroscopies, are currently reported to deliver a comprehensive set of data, at the conformational and dynamic levels, on: (i) NTC transformation [6]; (ii) activity of newly developed Pt/Pd-anticancer agents (on DNA, glutathione, proteins, cellular metabolism and intracellular water) [7-9]. Variations in the dynamical profile of intracellular water were unveiled for malignant cells/tissues as compared to healthy ones. In addition, clearly distinct effects were revealed for Pt- vs Pd-agents regarding their impact on either the cellular cytoplasm or hydration water in cancer cells, as well as concerning their specific interactions with biomolecules. This is a pioneer study on the impact of cisplatin-like hemotherapeutic agents on vital cellular components, which is key for a thorough understanding of their molecular basis of cytotoxicity.
These results are expected to foster the development of improved anticancer drugs -displaying high specificity and optimised efficacy. Ideally, these are aimed to act simultaneously on more than one site (multitarget approach), intracellular water being suggested as a potential pharmacological target. Advanced chemotherapeutic strategies such as these will contribute to a better prognosis and quality of life of cancer patients.

[1] N.P. Farrell Chem.Soc.Rev. 44 (2015) 8773. https://doi.org/10.1039/C5CS00201J
[2] M.P.M. Marques et al. J.Phys.Chem.B (2019) 123, 6968. https://doi.org/10.1021/acs.jpcb.9b05596
[3] L.A.E. Batista de Carvalho et al. Biophys.J. (2021), 3070. https://doi.org/10.1016/j.bpj.2021.06.012
[4] R.C. Laginha et al. Int.J.Mol.Sci. (2023) 24, 1888. https://www.mdpi.com/14220067/24/3/1888/pdf)
[5] M.P.M. Marques et al. ISRN Spectroscopy (2013) 2013, 287353. https://doi.org/10.1155/2013/287353
[6] M.P.M. Marques et al. Sci.Rep. (2023) 13, 21079. https://doi.org/10.1038/s41598-023-47649-w
[7] M.P.M. Marques et al. Molecules (2020) 25, Article 246. https://doi.org/10.3390/molecules25020246
[8] M.P.M. Marques et al. Int.Rev.Phys.Chem. (2020) 39, 67. https://doi.org/10.1080/0144235X.2020.1700083
[9] M.P.M. Marques et al. PhysChemChemPhys (2022) 24, 15406. https://doi.org/10.1039/D2CP00621A)

Speaker: Prof. Maria Paula Marques (University of Coimbra)

Maria_EBSA25.pptx

12:00 PM TBD - Deckert

Speaker: Prof. Volker Deckert (Friedrich-Schiller University)

12:35 PM Nanospectroscopy Study of Amyloid Aggregates Interacting with RNA

Studying structural changes associated with protein aggregation is challenging and often requires a combination of experimental techniques to capture insights at the molecular level across different scales, from nanometers to microns. Studying this process becomes even more complex when aggregation occurs in the presence of molecular co-factors, nucleic acids among them, and when the resulting aggregates exhibit a high structural and morphological polymorphism. Here, we investigate the potential structural effects of RNA on amyloid protein fibrils. To achieve this, infrared (IR) spectroscopy, known for its high sensitivity to structural changes in the cross-β architecture of protein aggregates, was employed. In particular, IR spectroscopic analysis was performed by combining Fourier transform infrared (FTIR) microspectroscopy (micro-FTIR) and IR nanospectroscopy approaches relying on the use of an atomic force microscope (AFM) to probe the supramolecular architecture of aggregates at the nanoscale. Co-incubation with RNA was shown to alter the α-synuclein (α-syn) fibril architecture by promoting the formation of more rigid fibrils and to reduce the structural polymorphism within the fibril population. Additionally, AFM morphological characterization on individual α-syn fibrils demonstrated that RNA modifies the morphological properties of fibrils, reducing their diameter and increasing their persistence length. Remarkably, IR nanospectroscopy experiments demonstrated that RNA had a more pronounced impact on the supramolecular architecture of α-syn ordered fibrils compared to less ordered amyloid aggregates, suggesting that RNA has distinct structural effects depending on the aggregate architecture. This finding suggests that RNA may have varying interaction affinities for different types of aggregates, leading to distinct modifications in their supramolecular architectures depending on their structural organization.

Speaker: Dr Antonia Intze (Physics Department, Sapienza University of Rome)

11:00 AM → 11:25 AM Coffee break: coffee break

12:55 PM → 2:10 PM Lunch

2:10 PM → 6:25 PM Session 2

2:10 PM FEL Coulomb Explosion Imaging: Simulation of Coulomb explosion of highly charged 2-Iodopyridine and comparison to experimental data

X-ray Single Particle Imaging has the aim of imaging biomolecules without the need of crystallization. The invention of X-ray Free Electron Lasers (XFELs) provided the instruments for this imaging process, however the technique is still suffering from low signal to noise ratio. Retrieval of the orientation of the sample in the moment of photon-sample interaction would greatly improve the signal and interpretability of experimental data. During the interaction, the intense radiation of the XFEL leads to high ionization of the sample and to a Coulomb explosion, in which positively charged ions repel each other and fly out of the interaction zone. It was found in recent simulations that a record of the Coulomb explosion doesn’t only just retrieve the original orientation of the imaged sample, but also bears structural information itself about the molecule. Hence, it was proposed to explore the relationship between structure and Coulomb explosion, a process called Coulomb Explosion Imaging. The biophysics research group of Uppsala University has developed a Molecular Dynamics/Monte Carlo code called MolDStruct4, which is based on GROMACS. MolDStruct replicates the radiation-induced Coulomb explosion of biomolecules and enables the tracking of ion-trajectories. The code is currently benchmarked against the QM/DFT code SIESTA as well as experimental data to ensure that the simulations of the Coulomb explosions are as realistic as possible. In this process, we investigate the biomolecule 2-Iodopyridine, ionize it up to an average ionization of one per atom and record the Coulomb explosion in simulations with MolDStruct. Then, we compare the results with data from a recent XFEL experiment in which highly ionized 2-Iodopyridine was exploded and the ejected ions were measured using a reaction microscope. Additionally, we execute QM/DFT simulations with the code SIESTA and also record the Coulomb explosion. The simulations with MolDStruct and SIESTA are consistent with one-another and they both replicate well the experimental data. Contrarily to Single Particle Imaging, Coulomb Explosion Imaging does not necessarily need hard X-rays and would be feasible with an intense FEL, such as the one being developed in the EUPRAXIA project. With MolDStruct, the relationship between structural information and Coulomb explosions can be studied intensively in simulations, information which can later on be used to support and enhance experiments on molecular sample structures by adding new imaging possibilities and providing better interpretation of the signal.

Speaker: Lais Friederike Krüger (Uppsala Unversitet)

2:15 PM Raman microscopy as a tool to investigate the effects of polystyrene nanoparticle in Zebrafish and human Caco-2 Cells

Marine microplastic pollution has emerged as a major global concern, with growing implications for both marine ecosystems and human health [1]. Polystyrene nanoparticles (PS-NPs) can induce significant biological responses, as demonstrated by combined in vivo and in vitro models. In zebrafish, exposure to PS-NPs resulted in marked changes in eye pigmentation patterns that were not associated with a reduction in melanin content or tyrosinase activity. In this context, Raman microscopy revealed structural differences between treated and untreated samples, possibly related to melanogenesis-inflammatory processes and oxidative stress - an interpretation supported by gene expression data showing strong upregulation of inflammation- and oxidative stress-related markers. In particular, increased expression of rpe65c, a gene associated with retinal health, suggests early signs of retinal dysfunction.
In parallel, human colorectal adenocarcinoma cells (Caco-2), used as an in vitro epithelial model, show efficient internalization of PS-NPs, with Principal Component Analysis (PCA) of Raman mapping data revealing a predominant cytoplasmic and perinuclear localization. Morphological changes consistent with cell death were also observed, indicating a dose- and time-dependent cytotoxic effect. These findings highlight the potential of micro-Raman mapping to reveal the effects of PS-NPs in biological samples, showing their disruptive effects on both developmental and cellular processes, raising concerns about their impact on ocular physiology and epithelial integrity.

Speaker: Mr Federico Perrella (Università degli Studi dell'Aquila)

2:20 PM Real-time observation of conical intersections in biomolecules.

Speaker: Prof. Giulio Cerullo (Politecnico di Milano)

2:55 PM Shedding Light on SARS-CoV-2 viral protein: Infrared Spectroscopy of the Receptor Binding Domain to Spike Protein

Proteins, constituting the virus structure, cover a wide and diverse range of functions. Spike glycoprotein (S) of SARS-CoV-2 is a notable example. As the largest structural protein of the virus, the S protein plays a crucial role in attaching to the host receptor ACE2 through its receptor-binding domain (RBD) [1]. The functionalities of these membrane proteins, such as cellular targeting and recognition, transport, and communication are affected by viral and host factors, including immune evasion, conformational masking of binding domains, glycan shielding, as well as the extent of receptor binding affinity and specificity [1,2].
Understanding the secondary structure of the S protein is crucial for gaining insights into its functionality and into the mechanisms occurring in the viral process, and for addressing specific actions aimed at developing specific drugs, diagnostic tools, and prevention strategies. In this context, vibrational spectroscopy, including infrared (IR) spectroscopy, offers various advantages: it is label-free, fast, non-contact and non-destructive, and it allows multi-component assays. In addition, IR frequency region examines localized molecular vibrations of macromolecules, such as carbohydrates, lipids, DNA and RNA, proteins and their mechanisms of reactions, processes like folding, unfolding, and misfolding, and their secondary structures [3,4].
Here, we present an overview of our results obtained from a systematic and comparative study of SARS-CoV-2 viral protein, its individual protein domains, namely the RBD, S1, S2 regions, and S protein, as well as SARS-CoV-2 S1 variants at serological pH, by measuring the amide I absorption band (1600-1700 cm-1) using Attenuated Total Reflection Infrared (ATR-IR) spectroscopy [5-7]. The combination of experimental results with predictive and computational approaches, such as Define Secondary Structure of Proteins (DSSP) predictions, Molecular Dynamics (MD) simulations and protein Surface Polarity Calculations, provides a comprehensive understanding of the protein domains in terms of their secondary structure content, 3D conformation, and interaction with the solvent.

References
[1] W.T. Harvey et al. Nat. Rev. Microbiol. 19, 409–424 (2021).
[2] R. Zhu et al. Nat. Commun. 13, 7926 (2022).
[3] A. Barth. Prog. Biophys. Mol. Biol. 74, 141–173 (2000).
[4] F. Piccirilli et. al. Nanomaterials 11(5), 1103 (2021).
[5] A. D’Arco et al. Int. J. Mol. Sci. 24(11), 9550 (2023).
[6] T. Mancini et al. Optical Sensors 12572, 58-62 (2023).
[7] T. Mancini et al. Adv. Sci., 11(39), 2400823 (2024).

Speaker: Annalisa D'Arco (Sapienza University of Rome, Dept. of Physics)

3:30 PM Low background high-repetition rate 3D X-ray imaging of single bio-particles using a helium-electrospray.

Imaging the structure and observing the dynamics of isolated proteins using single-particle X-ray diffractive imaging (SPI) is one of the potential applications of X-ray free-electron lasers (XFELs). Currently, SPI experiments on isolated proteins are limited by three factors: low signal strength, limited data and high background from gas scattering. The last two factors are largely due to the shortcomings of the aerosol sample delivery methods in use. Here we present our modified electrospray ionization (ESI) source, which we dubbed helium-ESI (He-ESI). With it, we decreased the gas load in the interaction chamber corresponding to an 80% reduction in gas scattering when compared to the original ES and increased particle delivery into the interaction region by a factor of 10, for 26 nm-sized biological particles. The increased particle delivery was measured using light scattering and also to measure the size and location of single viruses and protein complexes forming an aerosol beam. We were able to detect individual particles down to 16 nm in diameter. The primary purpose of our scattering instrument is to monitor the delivery of single bioparticles to the focus on an X-ray laser and using the He-ESI to potentially increase the quality and quantity of SPI diffraction patterns in future experiments resulting in higher-resolution structures.
In November 2023 we performed an SPI experiment at the SQS endstation on various samples based on low gas background gas scattering. We reduced the background from the gas scattering by a factor of ~ 5 and obtained many diffraction patterns from Bacteriophage MS2, a small virus. Most significantly, we have collected the first dataset of a protein complex, Photosystem I, an important membrane protein, by X-ray SPI. The recorded diffraction patterns match that of photosystem I and we estimated a resolution of 6 nm by phase retrieval transfer function.

Speaker: TEJ VARMA YENUPURI (UPPSALA UNIVERSITY)

3:50 PM Picosecond X-ray pulses at Elettra 2.0 with crab cavities

Picosecond-long x-ray pulses of moderate intensity and up to MHz CW repetition rate for time-resolved analysis of matter in the linear response regime are proposed for un upgrade operation of Elettra 2.0, now in construction as funded successor of the Elettra storage ring light source in Trieste, Italy. The scheme, based on the adoption of radiofrequency transverse deflecting cavities, promises a spectral flux at 1–10% level of the standard single bunch emission at the sample, transverse coherence in both transverse planes up to 0.5 keV photon energy, and it turns out to be simultaneous and largely transparent to the standard multi-bunch operation. The project well matches the view of an integration of storage ring and free electron laser communities, viewing their co-location as a crucial asset for advancing time-resolved science.

Speaker: Simone Di Mitri (Elettra Sincrotrone Trieste)

Di Mitri_BioMol2025.pptx

4:35 PM Single particle imaging of biomolecules using Coulomb Explosions.

Single particle imaging of biomolecules using Free electron lasers (FEL) is an imaging technique that has been under development since the dawn of FEL:s more than two decades ago. Due to the heavy ionisation, biomolecules exposed to FEL pulses explode. In a recent publication (Phys. Rev. Lett. 134, 128403 (2025)) we have described how we can harvest information about the molecular structure of protein, solely but measuring the ions ejected from the explosion. In this simulation study we were able to separate protein structures that have identical amino acid sequences, but slightly different folding. This study opens up a pathway where it would be possible to use photon sources with wavelength that traditionally would not be suitable for imaging, like the AQUA instrument at EuPRAXIA@SPARC_LAB.

Speaker: Carl Caleman (Uppsala University)

5:10 PM TBD - Scopigno

Speaker: Prof. Tullio Scopigno (University Rome La Sapienza)

5:45 PM Enhanching Radiotherapy with High-Z doped Nitroimidazoles

A way to induce local damage to cancerous tissue is by using radiotherapy-amplifying bioagents doped with high-Z elements. This enables deep core-level ionisation during radiotherapy with X-rays above the K-edge threshold, significantly increasing radiation absorption. Core electron ejection from high-Z elements also triggers a cascade of secondary particles, amplifying damage.
We studied the iodine- and bromine-doped nitroimidazole molecule, an oxygen mimetic that accumulates in oxygen-deficient tumours. We analysed fragmentation mechanisms and radiotherapy-relevant fragments in the gas phase using synchrotron light tuned to K- and L-edges. Additionally, DFT-based molecular dynamics simulations explored bond strengths and fragmentation pathways. To approximate biological conditions, we also examined monosolvated nitroimidazole.
High-Z ionisation produces large quantities of single-atom ions, while C, N, or O 1s-ionization yields heavier fragments like NO2, which can inhibit DNA repair. The addition of a single water molecule affects the local chemical environment and is thus reshaping the dissociation landscape, possibly through hydrogen bonding and charge redistribution—suggested to protect biomolecules from radiation damage.

Speaker: Pamela Svensson

6:05 PM Molecular dynamics simulations of large gas-phase proteins with the fast multipole method

Classical all-atom molecular dynamics (MD) simulations are a powerful tool for investigating the structures and interactions of biomolecules in the gas phase. Until recently, these simulations were limited to systems containing only a few thousand atoms, due to the quadratic scaling of computational cost with system size. However, the recent integration of a linearly-scaling algorithm for computing the long-range electrostatic forces—the fast multipole method (FMM)—into the MD engine GROMACS has significantly enhanced performance for large biomolecular systems. With this advancement, MD simulations can now be performed efficiently on systems in the megadalton (MDa) range, aligning more closely with the sizes of biomolecules investigated in XFEL experiments.

Here, we demonstrate how to effectively use FMM to achieve high accuracy and performance, and present insights from its initial application to several multimeric protein complexes. Extending the simulation time up to one microsecond, we have gained new insights about the behavior of large proteins in vacuum. Simulations were also performed at multiple temperatures to mimic the effects of thermal activation via collisions with inert gas. These collisions range from low-energy impacts commonly used during electrospray ionization to higher-energy impacts used in techniques like ion mobility spectrometry to probe structural rearrangements and conformational stability. Overall, we demonstrate that FMM-accelerated MD is a powerful tool for investigating large gas-phase biomolecules, yielding results directly relevant to a wide range of gas-phase experimental techniques.

Speaker: Louise J. Persson (Uppsala University)

4:10 PM → 4:35 PM Coffee break

7:30 PM → 10:00 PM Social dinner

Monday, June 30

9:30 AM → 12:45 PM Session 3

9:30 AM TBD - Bean

Speaker: Richard Bean

10:05 AM Sample delivery for time-resolved studies at the European XFEL

The European XFEL (EuXFEL) enables high resolution and time-resolved structural studies of biological systems. Diffraction, scattering and spectroscopy experiments with a MHz pulse repetition rate, in combination with optical pump-probe or mixing set-ups, give new insights into protein motion and dynamics. To accommodate the MHz repetition rate, sample delivery methods are in demand which ensure quick and efficient sample renewal. The Sample Environment and Characterization (SEC) group at EuXFEL develops these methods and supports users during their experiments. In this presentation sample delivery methods will be discussed and the SEC group activities will be described.

Speaker: Katerina Dörner

10:40 AM X-ray Emission Spectroscopy for Real-Time Diagnostics in SFX experiments: A Machine Learning Approach

X-ray emission spectroscopy (XES) complements structural techniques like serial femtosecond crystallography (SFX) by providing insights into the electronic states at specific sites within a sample. At X-ray free-electron lasers, simultaneous SFX and XES measurements using a single pulse have already been performed — such as the determination of the oxidation states in metalloproteins. By using non-thermal plasma simulations alongside relativistic atomic data, we trained a neural network on synthetic XES data from protein crystals to predict fluence and pulse duration of the beam. Trained on synthetic data generated by a collisional radiative model, the network accurately predicts fluence with <1.5% relative error and when predicting both parameters concurrently with <12% error. Feature importance analyses reveal spectral regions tied to underlying physical mechanisms. The model emphasizes K to L shifts to higher emission energies due to presence of highly charged sulfur ions. This approach performs comparably to, and in some cases better than, current experimental setups that rely mainly on upstream X-ray gas monitors (XGMs). It offers a promising route toward real-time, high-repetition-rate diagnostics, effectively complementing existing XFEL beam characterization methods.

Speaker: Alfredo Bellisario (Uppsala University)

11:25 AM TRISS: A Versatile Tool for Unraveling Molecular Mechanisms

TRISS (TRapped Ion Spectrometer Setup) is a novel experimental station at the MAX IV synchrotron facility, designed to investigate fundamental molecular processes relevant to biomolecular and biochemical physics. TRISS uses an electrospray ionization (ESI) source to create ions, which are then fragmented using photons, electrons, or gas. The TRISS setup combines a segmented linear ion trap (the Omnitrap) with time-of-flight mass spectrometry. The Omnitrap's design enables precise manipulation of ions, including their storage, isolation, and fragmentation. By providing detailed insights into how molecular structures break down, TRISS contributes to a deeper understanding of fundamental molecular interactions, with applications in areas such as radiation-induced damage.

Speaker: Ouassim Hocine Hafiani (Uppsala University)

11:30 AM Brute Force Orientation of Proteins

Diffractive single-particle imaging (SPI) using X-ray free-electron
lasers (XFELs) offers a promising approach for determining protein
structures without crystallization (Neutze2000) . For successful
reconstruction thousands of diffraction images of individual proteins
have to be assembled. It has been shown with molecular dynamics
simulations that proteins carrying a dipole moment can be oriented with
external electric fields (Marklund2017) and that the computer
algorithm that sorts the diffraction images benefits from
pre-orientation (Marklund2017, Wollter2024). A general estimate of
the required minimum field strengths for sufficient orientation is still
unknown.\
Expressions for the distribution of the angle $\theta$ between the
dipole moment $\mu$ and the field $\epsilon$ were derived for 1) a
theoretical mechanical rigid rotor model (RR) of a single protein in
which no energy is transferred to inner degrees of freedom and 2) a
theoretical thermodynamic model (TM) that assumes equilibration of all
internal degrees of freedom in the protein. Molecular dynamics (MD)
simulations show similarity with RR for low fields and good agreement
with TM for higher fields, when the simulation duration is sufficiently long
for the system to reach equilibration.

Based on RR for a single protein and assuming random orientation as well
as Boltzmann-distributed rotational kinetic energy when entering the
field region, we estimate the distribution of orientation angles for an
ensemble of proteins.

To study the beneficial effect of pre-orientation we performed Enhanced
EMC (Marklund2017, Wollter2024) with thousands of simulated
diffraction pattens (Hantke2016) of proteins oriented according
to the theoretical distributions (RR ensemble, TM).

We estimate the required fields for a given dipole moment to achieve
given angular confinements and for EEMC to benefit from pre-orientation
and relate them to the dipole moments of a dataset of 60k proteins
lankar2016).
We conclude that TM is suitable to describe the distribution of angles
even for a single protein and that field strengths required to achieve
sufficient orientation for EEMC to benefit are technically feasible for
a wide range of proteins.

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Speaker: Thomas Mandl (Uppsala universitet)

11:35 AM TBD - Weik

Speaker: Dr Martin Weik (Institut de Biologie Structurale)

12:10 PM Serial macromolecular crystallography: developments for high throughput measurements over large parameter spaces

Serial macromolecular crystallography (SMX) has emerged as a transformative technique enabling measurements across expansive parameter spaces in structural biology. Here I will talk about the evolution of serial crystallography techniques, particularly focusing on developments for time-resolved, pH-responsive, and temperature-controlled experiments and their application in unraveling dynamic structural changes within macromolecules. Highlighting recent results from X-ray Free Electron Laser (XFEL) and synchrotron sources, this presentation explores how serial crystallographyin in combination with cutting edge sample delivery and triggering methods can be used to advance our understanding of biological processes.

Speaker: Dr Dominik Oberthür (Center for Free-electron Laser Science)

11:00 AM → 11:25 AM Coffee break

12:45 PM → 2:00 PM Lunch

2:00 PM → 4:05 PM Session 4

2:00 PM TBD - Baranska

Speaker: Prof. Malgorzata Baranska (Jagiellonian University)

2:35 PM FTIR (micro-)spectroscopy in situ: Diagnostic Potential and Insights into Amyloid Deposits

Fourier transform infrared (FTIR) (micro-)spectroscopy is a label-free and non-destructive vibrational tool that has been successfully applied to study not only the amyloid structural properties and aggregation mechanisms directly in cells, tissues, or biofluids, but also to gain new insights into the mechanisms of amyloid formation and toxicity[1]. In particular, the use of an infrared microscope allows measuring in situ the IR absorption from selected areas of the sample, enabling to explore the colocalization of amyloid deposits with other biomolecules[1].
We employed FTIR microspectroscopy to analyze unfixed human tissues - cardiac and adipose - from patients affected by systemic light chain amyloidosis[2]. We detected the in situ marker band of the aggregates, ascribable to amyloid deposits. The possibility to measure unfixed tissue sections made it possible to detect important peculiarities in the spectral features of other biomolecules in cardiac tissues, in areas enriched with aggregates, suggesting a role in particular of lipids in amyloid deposition in vivo[2].
We then applied attenuated total reflection (ATR)-FTIR investigation - coupled to multivariate analysis - to the analysis of adipose tissue aspirates from patients affected by systemic amyloidosis3. We found that the ATR-FTIR approach can differentiate fat aspirates containing amyloid deposits from control specimens with high sensitivity and specificity. Notably, discrimination between amyloid-affected and negative samples was obtained on the basis of the whole spectrum, pointing out that resident lipids are intrinsic features of amyloidosis-affected subcutaneous fat[3].
After our initial studies[2,3], independent groups published works on the potential use of FTIR spectroscopy for the detection and typing of cardiac amyloidosis. The results of subsequent studies[4] confirmed our findings on a larger cohort of patients, emphasizing FTIR spectroscopy as a promising diagnostic method. Here, the scientific background[1,5] and the FTIR spectroscopic approach[1-3] are presented for the detection of cardiac amyloidosis in a clinical setting on the one hand, and for gaining new insights into amyloid deposits in situ on the other.

References:

1. Ami et al. Front Mol Biosci. 2022;9:822852. doi: 10.3389/fmolb.2022.822852.
2. Ami et al. Sci Rep. 2016, 6:29096. doi: 10.1038/srep29096.
3. Ami et al. Anal Chem. 2019; 91(4):2894-2900. doi: 10.1021/acs.analchem.8b05008.
4. Mukherjee et al. Circulation. 2024, 150(16):1299-1301. doi: 10.1161/CIRCULATIONAHA.124.069554.
5. Lavatelli et al. J Biol Chem. 2024;300(4):107174. doi: 10.1016/j.jbc.2024.107174

Speaker: Antonino Natalello (Dept. of Biotechnology and Biosciences, University of Milano-Bicocca, Milano (Italy))

2:55 PM Multidimensional IR Spectroscopy (2D-IR) as tool to study protein dynamics

Ultrafast multidimensional IR spectroscopy (2D-IR) can provide detailed information on local structural dynamics in peptide, proteins or complex systems like models of protein condensates. 2D-IR is a femtosecond laser spectroscopy with its intrinsic time-resolution on par with the fundamental timescale of chemical dynamics, fs to ps. For biological systems the capability to directly probe H-bonding dynamics, changes of electrostatics and conformational dynamics are important. In particular for intrinsically disordered systems or beta-sheet rich structures, 2D-IR has benefits over FTIR spectroscopy, as weak signals (i.e. shoulders) are much sharper in the 2D-IR spectra. The information obtained from 2D-IR spectra is thus complementary to insights from X-Ray diffraction experiments. In this contribution, the basics of 2D-IR and applications to biomolecular systems shall be reviewed and prospects for future integration between 2D-IR and XFEL experiments will be addressed. To this end, we have already performed initial 2D-IR experiments on protein crystals with fixed target sample delivery similar as for TR-SFX experiments.

Speaker: Henrike Müller-Werkmeister (University of Potsdam)

3:15 PM Round Table and concluding remarks