# 11th International Workshop on X-ray Radiation Damage to Biological Samples - RD11

Europe/Zurich
WBGB/019 (Paul Scherrer Institut)

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
Description

The Workshop will address the essential questions and challenges of radiation damage to biological samples during their examination with ionizing radiation. The workshop will cover various X-ray and electron scattering techniques, from crystallography to imaging, and offers ample opportunities for information exchange and discussion among researchers from around the globe.

Sessions will cover:

1. Biological Studies Affected by Radiation Damage
2. Practical Aspects of Reducing Radiation Damage at Synchrotrons
3. Damage at New Sources - XFEL and 4th generation synchrotrons
4. Radiation Damage in Complementary Fields (incl. CryoEM, ED, imaging)
Contact
• Wednesday, 25 March
• 11:30 12:00
General: Registration opens WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 12:00 13:00
Lunch 1h WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 13:00 13:20
General: Welcome WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 13:20 15:50
Session 1 - Biological Studies Affected by Radiation Damage WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 13:20
Approaches for following X-ray induced reactions in crystallo 20m

Robin Owen1, Danny Axford1, Ali Ebrahim1,2, Selina Storm1, Hiroshi Sugimoto3, Kensuke Tono3, Shigeki Owada3 Tadeo Moreno-Chicano2, Richard Strange2, Jonathan Worrall2 , Ivo Tews3 & Michael Hough2

1Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, UK
2School of life Sciences, University of Essex, Wivenhoe Park, Colchester, Essex, CO4 3SQ, UK
3Spring8/SACLA, Harima Campus, Hyogo, Japan.
4Biological Sciences, University of Southampton, Institute for Life Sciences, Southampton, SO17 1 BJ, UK

robin.owen@diamond.ac.uk

The development of serial crystallography has been driven by XFELs but is now firmly established at high-brilliance synchrotron sources. Serial crystallography provides a means of efficiently collecting diffraction data from crystals held at ambient temperatures, providing access to more physiological environments and helping make structural transitions directly observable. Data collection at room temperature comes, however, with a price in the form of the rapid onset of radiation damage and greatly reduced crystal lifetimes. While this drawback can be addressed in part through the use of serial approaches, significant challenges for the experimenter remain.
I will describe fixed target serial delivery methods developed and implemented at SACLA and Diamond, illustrating the gains that can be realised using a multi-faceted approach at different, complementary, sources as well as some of the challenges both met and remaining.

Speaker: Dr Robin Owen (Diamond Light Source)
• 13:50
X-ray induced radiation damaged on metal centers in proteins 20m

CAN ONLY SPEAK ON DAY 1

Speaker: Kristina Carugo
• 14:20
X-ray free electron lasers reveal the molecular mechanism for water oxidation in photosystem II 20m

Photosynthetic water oxidation is catalyzed by the Mn4CaO5-cluster1,2 of Photosystem II (PSII) through a linear four oxidation intermediates of Si-state cycle (Si, i = 0-4). The catalyst becomes a Mn4CaO6-cluster in the S3-state by incorporation of additional oxygen O6 nearby a unique central oxo-bridge O53, supporting a dioxygen formation mechanism between O5 and O6. While insertion of the O6 has gradually been accepted, the chemical structure of O5 and O6 remain controversial so that several possible mechanisms for the O=O bond formation have been under debate.
To reveal the molecular details in the water oxidation reaction, we analyzed the X-ray free laser (XFEL) structures of PSII in the S1, S2 and S3 states by using fixed-target serial femtosecond crystallography with an XFEL provided by the SACLA4. In brief, single-shot diffraction images were collected in a fixed-data collection manner at a cryogenic temperature, in which PSII microcrystals were evenly sprayed on a mesh. Compared with serial femtosecond crystallography of PSII using a grease matrix as the injection medium at room temperature3, this method reduced the sample consumption by one order of magnitude and ensured low background images, allowing us to analyze multiple datasets at 2.15-Å resolutions.
No insertion of water was found in the S2 state, but upon transition to the S3 state, flipping of Glu-189 provides a space for incorporation of the additional oxygen O6, and the Mn4CaO5-cluster remains in the open-cubane form. To determine the exact chemical structure of the O5 and O6, we examined four possible chemical species; superoxo, peroxo, oxyl/oxo, and oxyl/hydroxo. By altering the O5-O6 distance and examining the residual densities in the Fobs ̶ Fcal difference Fourier map, we found that a distance of 1.9 Å resulted in the weakest residual densities. This suggests an oxyl/oxo coupling mechanism for the O=O bond formation in OEC. Moreover, the flipping of the Glu-189 also induces van der Waals repulsion between the carbonyl oxygen of Glu-189 and Ala-411CP43, which is transmitted to a strictly conserved short loop of the CP43 subunit that restricts the size of the O1-water channel. These structural changes in PSII between the different S-states reveal the mechanism of photosynthetic water oxidation by the cooperative action of substrate water access, proton release, and O=O bond formation.

Acknowledgments
We thank many collaborators who are not listed here due to the limited space.

References
1. Umena, Y. et. al. Nature 473, 55-60, (2011).
2. Suga, M. et al. Nature 517, 99-103, (2015).
3. Suga, M. et al. Nature 543, 131-135, (2017).
4. Suga, M. et al. Science 366, 334-338, (2019).

Speaker: Michi Suga (Okayama University)
• 14:50
Applications of Room Temperature Serial Crystallography to Metalloproteins using Silicon Fixed Targets at Synchrotron and XFEL beamlines 20m

Metalloproteins, particularly those containing transition metals in high valent states are highly sensitive to redox state changes as a consequence of X-ray exposure during data collection. If not under-stood and interpreted correctly, this site-specific damage may lead to misinterpretation of structural data and mis-assignment of structures to e.g. enzyme mechanistic states. Increasing attention has been paid to room temperature crystallography to allow for protein dynamic freedom and in order to facilitate time-resolved crystallography. The binding of ligands to proteins may be affected both by temperature and redox state. The faster rate of radiation damage with dose in room temperature crystals poses challenges for data collection methodologies. I will describe room temperature fixed target ‘chip’ serial synchrotron crystallography (SSX) measurements at Diamond beamline I24 and serial femtosecond crystallography (SFX) experiments at the SACLA X-ray Free Electron Laser (XFEL), Japan. This system may be implemented with few changes at either synchrotron micro-focus or XFEL beamlines allowing us to directly compare the effects of radiation damage in SSX structures to XFEL structures collected using a near identical strategy i.e. where the nature of the X-ray beam is the primary experimental variable. I will describe applications of this approach to several metalloprotein systems [1-3].

References
[1] Resolving polymorphs and radiation-driven effects in microcrystals using fixed-target serial synchrotron crystallography. Ebrahim, A. et al., (2018) Acta Cryst. D75, 151-159.
[2] Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins. Ebrahim, A. et al., (2018) IUCrJ 6, 543-551.
[3] High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography. Moreno-Chicano, T., et al. (2019) IUCrJ 6, 1074-1085.

Speaker: Dr Mike Hough (University of Essex)
• 15:20
X-ray footprinting of proteins: using hydroxyl radical damage to infer structural conformation 20m

The method of X-ray footprinting mass spectrometry (XFMS) is used to investigate structural features and conformational changes of macromolecules in the solution state. XFMS is an in situ hydroxyl radical (•OH) labeling method; X-ray irradiation dissociates solvent water to produce hydroxyl radicals, which covalently modify side chains which are solvent accessible. More specifically, residues which are in proximity to water molecules (either bulk or bound) are modified to a greater extent than residues which are not in proximity to water. Because liquid chromatography-mass spectrometry is then used to analyze the stable covalent modifications produced, the data provide a “water map” at the single residue level, which is then used to determine sample conformation. In this talk, I will describe the XFMS method, its advantages and disadvantages relative to other methods, some recent examples of structural information obtained on protein systems using the method, and some recent instrumentation advances and plans for future improvements to the method.

Speaker: Corie Ralston (Lawrence Berkeley National Laboratory)
• 15:50 16:15
Coffee Break 25m WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 16:15 17:15
Session 2 - Practical Aspects of Reducing Radiation Damage at Synchrotrons WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 16:15
Effects of dose control in multiple small wedge data collection 20m

Recent brilliant micro-focused X-ray has enabled high resolution structural analyses of challenging biological proteins such from 10-µm sized crystals. One of the key technologies to achieve this is merging many partial datasets. This so-called ‘multiple small wedge data collection’ strategy was initially developed at Advanced Photons Source to accomplish data collection from in-meso phase crystals of membrane proteins. The loop containing several tens of tiny LCP crystals is raster scanned by low dose micro-focused beam. Based on this scan, crystals embedded in lipids invisible under the visible light are aligned to X-rays. From the angle where raster scan is applied, small wedge dataset is collected from each found crystal. Finally, many wedges from many crystals are merged into the final dataset.
At SPring-8, we have achieved to determine many membrane protein structures in this strategy. Recently, we developed ZOO system which enables un-attended fully automated data collection. The system has accelerated data collection in multiple small wedge scheme. A function to estimate absorbed dose for data collection is equipped to the system. Our recommended absorbed dose for all data collection scheme is 10 MGy under a cryo condition. It is based on our knowledge-based parameter in study of radiation damage by using micro-focused beam. However, no body have conducted the systematic study of effect of accumulated absorbed dose in ‘multiple small wedge’ strategy. In the full rotation data collection using a single crystal without any translation of irradiation points, radiation damage heterogeneously accumulated on the crystal volume and effects to errors in intensities in the identical dataset. On the other hand, multiple small wedge data collection is based on merging many datasets. In this case, merging equilibrates the radiation damage because heavily damaged data region is slightly compensated by another crystal with freshness against to the damage.
We systematically investigated effects of dose control in multiple small wedge data collection by using several standard protein samples. We will share our results to have discussions about the best dose for small wedge data collection.

Speaker: Kunio Hirata (RIKEN)
• 16:45
DOZOR, MESH-BEST and 3D-BEST: software for data collection optimization taking into account radiation damage 20m

Success of crystallographic experiments using weakly diffracting macromolecular crystals depends on a proper choice of the data collection protocol. Radiation damage is a critical factor limiting data quality and being able to predict the absorbed dose and mitigate the effects of exposure to X-rays is very important.
We present the set of methods and programs intended for optimal planning of X-ray diffraction by accounting the experimental purpose, the characteristics of the X-ray beam and instrumentation and by the samples properties. The information about the crystals mounted in the sample holder is taken from low-dose two-dimensional raster X-ray diffraction scans. The image analysis is carried out by program Dozor which recognizes the presence of a diffraction pattern from a macromolecular crystal, estimates the diffraction signal and produces the list of diffraction spot positions. Bases on Dozor’s output, the program MeshBest determines the areas of the mesh-scans which belong to the individual crystals and produces a three-dimensional crystal map showing estimates of the dimensions, centre positions and diffraction qualities of each crystal contained in the holder. Using this map, the best achievable results of data collection for any individual crystals are estimated by software program 3D-BEST. In last version of BEST, the strategy-determination method takes into account the variations in irradiated crystal volume at the rotation and possible translation of the sample. Diffraction intensities at any moment of data collection is calculated as the sum of diffraction intensities from small crystal voxels taking into account the profile of primary X-ray beam and the dose absorbed by each voxel. Such modelling significantly improves the accuracy of predicted data statistics and allows selecting the best trajectory of the crystal movements for helical or for multi-positional strategy of data collection.

Speaker: Dr Alexander Popov (ESRF)
• 17:15 18:00
General: Open Discussion WBGB/019

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#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
Conveners: Florian Dworkowski (Paul Scherrer Institut), Ian Carmichael
• 18:00 19:00
Buffet Dinner 1h Time Out ()

### Time Out

• 19:10 19:30
Poster: Poster Blitz WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 19:30 21:00
Poster: Postersession and Aperó SLS Entrance Hall ()

### SLS Entrance Hall

• Thursday, 26 March
• 09:00 10:30
Session 3 - Damage at New Sources - XFEL and 4th generation synchrotrons WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 09:00
UV-induced cleavage and geminate recombination of the disulfide bond motif followed via ultrafast X-ray absorption spectroscopy 20m

We investigated photocleavage of the disulfide bond motif in model compounds such as cysteine dimer (L-cystine) by time-resolved X-ray absorption spectroscopy [1,2]. We follow changes in X-ray absorption at the sulfur K-edge (2.5 keV) that appear to be unique to thyil (R-S∙) radicals, thereby tracking the fate of the disulfide bond. Ultrafast spectroscopy has revealed homolytic S-S bond cleavage upon ultrafast 267-nm excitation [3], with a dominant yield of geminate recombination in solution within the first picosecond. A minor fraction of the thyil radicals survives at least for microseconds, reminiscent of X-ray induced radical formation [4]. Doubling the ultraviolet excitation energy yields new photoproducts which indicate C-S bond cleavage. Weak relaxation of thyil charge density favors geminate recombination, suggesting at a natural inhibition of radiation from mid-ultraviolet excitation.
[1] M. Ochmann et al., J. Am. Chem. Soc. 139, 4797 (2017)
[2] M. Ochmann et al., J. Am. Chem. Soc. 140, 6554 (2018)
[3] K. Schnorr et al., J. Phys. Chem. Lett. 10, 1382 (2019)
[4] E. Y. Sneeden et al., J. Am. Chem. Soc. 139, 11519 (2017)

Speaker: Nils Huse (U. Hamburg)
• 09:30
Structural dynamics in proteins induced by and probed with X-ray free-electron laser pulses 20m

X-ray free-electron lasers (XFELs) enable crystallographic structure determination beyond the limitations imposed upon synchrotron measurements by radiation damage. The need for very short XFEL pulses can be relieved through gating of Bragg diffraction by loss of crystalline order as damage progresses but not if ionization events are spatially non-uniform due to underlying elemental distributions, as in biological samples. Indeed, correlated movements of iron and sulphur ions were observed in XFEL-irradiated ferredoxin microcrystals using unusually long pulses of 80 fs. Here we report a femtosecond time-resolved X-ray pump/X-ray probe experiment on protein nanocrystals. We observe changes in the protein backbone and aromatic residues as well as disulphide bridges. Simulations show that the latter’s correlated structural dynamics are much slower than expected for the predicted high atomic charge states due to the significant impact of ion caging and plasma electron screening. This indicates that dense-environment effects can strongly affect local radiation damage-induced structural dynamics.

Speaker: Alexander Gorel (Max-Planck-Insitute for Medical Research Heidelberg)
• 10:00
Specific radiation damage is a lesser concern at room temperature 20m

There are two types of radiation damage in protein X-ray crystallography [1]. The first one, global damage, has been known since the beginning of X-ray crystallography. Global damage accounts for the decrease in the diffraction properties of a crystal during data collection due to the interaction of X-rays with the atoms of the crystal, which, in particular, generates free electrons and radicals in the bulk solvent, progressively destroying the crystalline order. Global damage is slowed down by roughly two orders of magnitude on the dose scale when the diffraction experiment is performed at cryogenic rather than at room temperature. Cryo-crystallography has led to the explosion in the number of crystallographic protein structures in the 1990’s as it allows determining a structure from a single crystal. However, it was then realized that a second type of radiation damage was at play in cryogenic experiments: specific damage. This damage affects certain specific chemical groups that are sensitive to electrons, for instance disulphide bonds, carboxylate groups or metal cations, which can be found in protein active sites. This can be explained by the fact that X-ray induced free electrons can still diffuse at cryogenic temperature. Therefore, specific damage may lead to artefacts in structural analysis of reaction intermediate states and thus in mechanistic interpretation. The discernibility of specific damage at cryogenic temperature means that there is a significant difference between the rates of the two types of damage, i.e. a ‘decoupling’ between the two phenomena. As room temperature protein crystallography is quickly developing thanks to the development of faster, noiseless detectors, of improved sample environment at room temperature and of the concept of serial crystallography, the question of the comparison of the respective rates of specific and global damage build-up at room temperature has become a hot topic. We have compared the rates of both damage build-up at cryogenic and room temperature for various proteins, including the reaction intermediate state of a fragment of a photoreceptor [2]. While the two types of damage are largely decoupled at cryogenic temperature (decoupling factor between 12 and 1600), they occur on a similar dose scale at room temperature (decoupling factor between 1 and 8). This indicates that depending on the studied protein, specific damage may not be a primary concern in crystallographic structure determination at room temperature, provided diffraction data can be collected from a single crystal. This should stimulate the development of time-resolved crystallography experiments at synchrotrons.

[1] Holton (2009) ‘A beginner's guide to radiation damage’ J. Synchrotron Rad. 16, 133–142.
[2] Gotthard et al. (2019). ‘Specific radiation damage is a lesser concern at room temperature’ IUCrJ, 6, 665-680.

Speaker: Dr Antoine Royant (ESRF)
• 10:30 10:55
Coffee Break 25m WBGB/019

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#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 10:55 12:25
Session 3 - Damage at New Sources - XFEL and 4th generation synchrotrons WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 10:55
Radiation damage in serial synchrotron crystallography at cryo- and room temperature 20m

References
[1] de la Mora E, Coquelle N, Bury CS, Rosenthal M, Holton JM, Carmichael I, Garman EF, Burghammer M, Colletier J-P, Weik M (2020) Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures. Proceedings of the National Academy of Sciences: 201821522

Speaker: Nicolas Coquelle (University Grenoble Alpes)
• 11:25
Correcting Non-isomorphism 20m

The problem of non-isomorphism has plagued macromolecular crystallography since the beginning [1, 2, 3], and it is essentially unavoidable in radiation damage studies. The unit cell changes with dose and that means the molecules in the cell must be adjusting somehow to the new cell. This will change the structure factors, but what if the molecular distortions could be corrected? Rigid-body motions cannot be the whole story because these lead to steric clashes. The true underlying distortion of the molecule must be both smooth across space, and also obey crystallographic symmetry. Here I present how periodic rubber-like distortions may be modelled using a collection of sine waves in space. This spatial distortion field (SDF) is similar in mathematical form to the Fourier synthesis of electron density from structure factors. The main differences are that the SDF is not a scalar field but a vector field describing changes in atomic position at every point in the unit cell. The number of orders needed to describe typical non-isomorphism is small, usually only 3-5 orders. SDFs may also be applied to electron density, allowing multi-crystal averaging across non-isomorphous crystal forms. Structural flexibility inherent to function may also be excited by these rubber-like distortions, making SDFs a potentially useful tool for elucidating subtle changes by eliminating the “noise” of rubber-like non-isomorphous distortion.

References
[1] B.S. Magdoff, F.H.C. Crick, Acta Cryst., 8 461 (1955).
[2] F.H.C. Crick, B.S. Magdoff, Acta Cryst., 9 901 (1956).
[3] C.C.F. Blake, R.H. Fenn, L.N. Johnson, D.F. Koenig, G.A. Mair, A.C.T. North, J.W.H. Oldham, D.C. Phillips, R.J. Poljak, V.R. Sarma, C.A. Vernon, in: M.G. Rossmann, E. Arnold (Eds.) International Tables for Crystallography F: Crystallography of biological macromolecules, Springer Netherlands, 2001. 745.

Speaker: Dr James M. Holton (University of California, San Francisco)
• 12:25 13:40
Buffet Lunch 1h 15m SLS Entrance Hall ()

### SLS Entrance Hall

• 13:40 15:10
Session 3 - Damage at New Sources - XFEL and 4th generation synchrotrons WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 13:40
Avoiding multiphoton artefacts in time-resolved pump probe experiments 20m

Light is important for organisms from all domains of life, serving as an energy resource or carrier of information initiating intra- or intercellular signaling. Photosensitive proteins, endowed with a light-absorbing chromophore, enable this. Obtaining direct structural information to understand the underlying molecular mechanisms is not only important for the fundamental understanding of light-driven processes but has practical impact on future developments of e.g. fluorescent proteins for optical nanoscopy or retinal proteins for optogenetics.

For time-resolved crystallography small crystals that can be efficiently photolyzed are required. Serial data collection by means of free-jet sample injection is a convenient way for time-resolved pump probe experiments. In previous experiments employing this technique both at X-ray free-electron laser (XFEL) and synchrotron sources very high pump laser fluences (≫ 100 µJ/mm²) have been used for optical excitation, a regime where multiphoton absorption of the chromophore is highly likely. However, multiphoton excitation differs significantly from the biologically relevant single photon absorption regime and generally results in different effects. This severely compromises the mechanistic interpretation.

We discuss this issue and present how to determine the appropriate photoexcitation intensity for time-resolved pump probe experiments. We discuss how to derive the required pump fluence based on the intensity distribution within the crystals and the jet used for delivering them into the X-ray beam and quantify losses in excitation intensity due to light-matter interaction (scattering, absorption, reflection) of the incident beam before reaching the target chromophores.

Speaker: Marie Luise Gruenbein (Max-Planck-Institute for Medical Research)
• 14:10
Is radiation damage the limiting factor in high-resolution single particle imaging with X-ray free-electron lasers? 20m

The prospect of single particle imaging with atomic resolution is one of the scientific drivers for the development of X-ray free-electron lasers. The assumption since the beginning has been that damage to the sample caused by intense X-ray pulses is one of the limiting factors for achieving subnanometer X-ray imaging of single particles and that X-ray pulses need to be as short as possible. Based on the molecular dynamics simulations of proteins in X-ray fields, we show that the noise in the diffracted signal caused by radiation damage is less than what can be expected from other sources, such as sample inhomogeneity and X-ray shot-to-shot variations. These findings show a different aspect of the feasibility of high-resolution single particle imaging using free-electron lasers, where employing X-ray pulses of longer durations could still provide a useful diffraction signal above the noise due to the Coulomb explosion.

Speaker: Carl Caleman (Uppsala University)
• 14:40
X-ray Emission Spectroscopy at X-ray Free Electron Lasers: Limits to Observation of Unperturbed Electronic Structures. 20m

Modern free electron lasers provide intense X-ray pulses with ~1012 photons within ~10-100 femtoseconds. Such pulses enable new experimental techniques and provide unique opportunities for investigation of electronic and nuclear dynamics on their intrinsic time-scales. Interaction of ultra-bright, ultra-short X-ray pulses with matter can induce a multitude of nonlinear excitation processes which must be carefully considered when planning spectroscopic measurements and interpreting data recorded at XFELs. In most cases correct interpretation of the spectroscopic response and analysis of the electronic structure hinges on the assumption of single photon excitations. Here we attempted to answer the fundamental question on the limits to probing the ground (or native) electronic structure of a 3d transition metal ion at XEFL sources. Ions of the 3d transition metal (Mn2+) in a lighter element (O, C, H) environment were used as a model system. X-ray emission spectroscopy recorded from Mn2+ at different pulse conditions demonstrate spectral changes as a function of increased pulse intensity and pulse duration. To explain these changes, we develop a rate equation based on sequential ionization and relaxation events forming multiply ionized states during a single pulse which agree with observed spectroscopic trends. The percentage of Mn Kβ emission recorded after the 1st, 2nd and 3rd 1s ionization events is calculated from the developed rate equation model and validated by experimental measurements. A method for estimating shifts in atomic X-ray emission lines from sequential ionization during a single XFEL pulse is given. From our data we infer that, in addition to multiple ionization, the impact of electron cascades is more significant for longer pulses. We note that while use of shorter X-ray pulses will help to counteract additional effects of electron cascades it will not help to overcome the spectral shifts due to sequential ionization. Presented data and associated analysis will help with experimental designs at current and upcoming XFELs where even higher intensities and shorter pulses are expected. 3d elements have a variety of important applications such as in bio-inorganic catalysis, chemical catalysis and energy storage / conversion making robust protocols for their XFEL analysis of general importance.

Speaker: Dr Yulia Pushkar
• 15:10 15:55
General: Open Discussion WBGB/019

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#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
Conveners: Arwen Pearson (Astbury Centre for Structural Molecular Biology, University of Leeds), Masaki Yamamoto (RIKEN/SPring-8 Center)
• 15:55 16:20
Coffee Break 25m WBGB/019

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#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 16:20 18:00
General: SLS/SwissFEL Tour WBGB/019

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#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 18:00 18:30
General: Walk to DInner WBGB/019

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#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 18:30 21:30
General: Conference DInner
• Friday, 27 March
• 09:00 10:30
Session 4 - Radiation Damage in Complementary Fields including Biological Imaging WBGB/019

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#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 09:00
Dose calculations for microcrystallography, XFELs, and electron microscopy: extensions to RADDOSE-3D 20m

Radiation damage remains a fundamental limitation to the success of X-ray macromolecular crystallography (MX) experiments. The program RADDOSE-3D [1,2] estimates the dose absorbed by samples during data collection at synchrotron sources, allowing direct comparison of radiation damage between experiments carried out with different samples and beam parameters. Here, I present a number of extensions to RADDOSE-3D, which perform Monte Carlo simulations to improve the accuracy and applicability of RADDOSE-3D. The first of these extensions provides more accurate dose estimates for synchrotron data collection on microbeams or microcrystals by taking into account the redistribution of photoelectrons produced both in the crystal and the surrounding. These emphasise the importance of beam energy [3,4,5], surrounding material, and crystal orientation on radiation damage to microcrystals. The second extension, RADDOSE-XFEL, calculates the time-resolved dose during XFEL data collection. The final extension, RADDOSE-EM, calculates the dose absorbed during single particle electron cryomicroscopy and micro-electron diffraction (MicroED) data collection. It is hoped that these extensions can be used to facilitate the study of radiation damage in new experiments and be used to maximise data collection efficiency.

1. Zeldin, O. B., Gerstel, M. & Garman, E. F. RADDOSE-3D: Time- and space-resolved modelling of dose in macromolecular crystallography. J. Appl. Crystallogr. 46, 1225–1230 (2013).
2. Bury, C. S., Brooks-Bartlett, J. C., Walsh, S. P. & Garman, E. F. Estimate your dose: RADDOSE-3D. Protein Sci. 27, 217–228 (2018).
3. Cowan, J. A. & Nave, C. The optimum conditions to collect X-ray data from very small samples. J. Synchrotron Radiat. 15, 458–462 (2008).
4. Dickerson, J. L. & Garman, E. F. The potential benefits of using higher X-ray energies for macromolecular crystallography. J. Synchrotron Radiat.
5. Storm, S. L. S. et al. Measuring energy-dependent photoelectron escape in microcrystals. IUCrJ 7, 129–135 (2020).
Speaker: Joshua Dickerson (MRC-LMB Cambridge)
• 09:30
Quantitative simulation tools for predicting radiation damage driven by high-intensity x-ray pulses 20m

One of the key opportunities offered by the development of x-ray free-electron lasers is the determination, at atomic resolution, of the three-dimensional structure of biologically relevant macromolecules. The basic idea underlying molecular imaging using x-ray free-electron lasers is the diffract-and-destroy'' concept: If one uses an x-ray pulse that is sufficiently short (on the order of femtoseconds), then in a single shot an x-ray scattering pattern may be obtained that is practically unaffected by atomic displacements triggered by ionization events during the x-ray pulse. What cannot be eliminated in this way is the impact of the electronic damage on the x-ray scattering patterns. Theory, therefore, plays an important role in the development of this new imaging technique: A quantitative understanding is required of the damage processes occurring during the exposure of a molecule to an ultraintense, ultrafast x-ray pulse. In this talk, I will present progress we have made in order to address this challenge. One tool we have developed, XMDYN, is a molecular-dynamics code that utilizes ab-initio atomic electronic-structure information, computed on the fly, within a Monte-Carlo framework. XMDYN has been successfully tested through experiments at LCLS and SACLA. XMDYN is part of a powerful start-to-end simulation framework for single-particle imaging at the European XFEL. Recently, we have taken first steps towards a full ab-initio framework for simulating high-intensity x-ray–matter interactions. Our new XMOLECULE software solves the polyatomic quantum-mechanical electronic-structure problem for every electronic state arising during the exposure of a molecule to a strong x-ray pulse. From this information, electronic transition rates (such as Auger decay rates) are computed on the fly, and the associated rate equations are integrated utilizing a Monte-Carlo method. XMOLECULE played a key role in a recent LCLS experiment on iodomethane, in which hard x-rays focused to a peak intensity exceeding $10^{19}$ W/cm$^2$ produced the highest charge states ever formed using light. Not only did XMOLECULE correctly predict the charge-state distribution observed, but it also helped identify a new molecular ionization enhancement mechanism based on intramolecular charge transfer.

Speaker: Robin Santra (DESY & Universität Hamburg)
• 10:00
Radiation damage in X-ray spectroscopy 20m

X-ray spectroscopy probes the electronic structure around an analyte element in a sample. The electronic structure relates to the formal oxidation and spin state and the atomic structure around the analyte element. The sensitivity to small modification of the electronic structure makes X-ray spectroscopy strongly responsive to small changes of the sample due to X-ray irradiation. Thus, X-ray spectroscopy often tolerates lower doses than atomic structural probes such as crystallography. The effect of X-rays in the sample may be oxidation or reduction of a metal site accompanied by modification of the local atomic coordination. Often, X-rays induce changes similar to other external triggers such as optical/UV illumination or temperature. This can be used to understand the changes in the sample, i.e. X-rays serve as source and probe of the sample modification. Radiation damage dramatically changes the experimental protocol to record spectroscopic data and some measurements become impossible. The presentation provides examples and discusses some fundamental concepts.

Speaker: Dr Pieter Glatzel (ESRF)
• 10:30 11:00
Coffee Break 30m WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 11:00 12:00
Session 4 - Radiation Damage in Complementary Fields including Biological Imaging WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
• 11:00
An Objective Metric for Correcting Radiation Damage in SAXS 20m

Size-exclusion chromatography small-angle X-ray solution scattering (SEC-SAXS) has become a standard method for modern bio-SAXS synchrotron light sources (Ryan et al. 2018; Brennich et al. 2016; Blanchet et al. 2015). However, the contamination of data by radiation damage remains a major issue resulting in poor post-processing of SEC-SAXS data. Several statistical tools have been developed to assess solution SAXS data quality (Rambo and Tainer 2013; Franke et al. 2015). These are mostly useful for “rejecting” significantly different data points or data frames, based on the assumption that the rest of the data are close to the “truth”. But this can lead to a situation where mediocre data, especially data contaminated with radiation damage, are not correctable or simply cannot be evaluated before any interpretation is done.

To alleviate this problem, an objective metric, correction-state score (CSS) based on the dynamic time warping algorithm (DTW) is proposed. CSS can be used to both verify the data quality and identify the optimal data correction procedure for post-processing of SEC-SAXS data. CSS can be represented as a numerical likelihood with a scale of 0 to 1. Using this objective score it is possible to quantitatively assess the “goodness” or appropriateness of a background correction for SEC-SAXS data and hence maximise the quality of the correction of radiation damaged datasets.

References
Blanchet, C.E., Spilotros, A., Schwemmer, F., et al. 2015. Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY). Journal of Applied Crystallography 48(Pt 2), pp. 431–443.
Brennich, M.E., Kieffer, J., Bonamis, G., et al. 2016. Online data analysis at the ESRF bioSAXS beamline, BM29. Journal of Applied Crystallography 49(1), pp. 203–212.
Franke, D., Jeffries, C.M. and Svergun, D.I. 2015. Correlation Map, a goodness-of-fit test for one-dimensional X-ray scattering spectra. Nature Methods 12(5), pp. 419–422.
Rambo, R.P. and Tainer, J.A. 2013. Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496(7446), pp. 477–481.
Ryan, T.M., Trewhella, J., Murphy, J.M., et al. 2018. An optimized SEC-SAXS system enabling high X-ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis. Journal of Applied Crystallography 51(1), pp. 97–111.

Speaker: Mr Yunyun Gao (Universität Hamburg/ Max-Planck Institute for Structure and Dynamics of Matter)
• 11:30
Domain swapping in solution induced by irradiation – case study human cystatin C and other proteins 20m

During small angle X-ray scattering studies of proteins in solutions, radiation damages may occur during exposure to the synchrotron radiation, which most often involves the protein aggregation process, but global conformational changes such as the domain swapping phenomenon is also possible.
The domain swapping phenomenon has been known for several decades and has been identified in numerous protein structures [1]. Particularly was observed for proteins of flexible structure. The phenomenon of domain swapping also accompanies of the amyloidogenesis process. This situation applies also to human cystatin C, where dimerization and oligomerization of this protein occurs through domain swapping [2,3].
In our previous studies, we observed domain swapping in the crystal structure of human cystatin C, where it probably occurred during the crystallization process. Recently we also observed, that this phenomenon for human cystatin C also occurs in solution and is caused by irradiation during SAXS experiments using synchrotron radiation [4]. During the lecture it will be discussed the radiation induced domain swapping of human cystatin C and other proteins.

[1] Liu, Y., Eisenberg, D. Protein Sci. 11(6), 1285-1299 (2002).
[2] Janowski, R. Kozak, M., Jankowska, E. Grzonka, Z. Abrahamson, M., Grubb, A. & Jaskolski, M. Nature Struct. Mol. Biol. 8, 316–320 (2001).
[3] Janowski, R., Kozak, M., Abrahamson, M., Grubb, A. & Jaskolski, M. Proteins: Struct. Funct., Bioinf. 61, 570–578 (2005).
[4] Taube, M., Pietralik, Z., Szymanska, A., Szutkowski, K., Clemens, D., Grubb, A., Kozak, M. Sci Rep 9, 8548 (2019).

Speaker: Maciej Kozak (Adam Mickiewicz University)
• 12:00 12:45
General: Discussion and Farewell WBGB/019

### WBGB/019

#### Paul Scherrer Institut

Forschungsstrasse 111 5232 Villigen PSI Switzerland
Conveners: Martin Weik (IBS), Vincent Olieric (PSI - Paul Scherrer Institut)
• 12:45 14:00
Lunch 1h 15m SLS Entrance Hall ()