Oral Presentation Crystal36-AXAA Conference 2026

Benchmarking fluctuation X-ray scattering as an indexing-free extension of serial crystallography (145031)

Alan G Salek 1 , Christopher Szeto 2 , Spencer Passmore 2 , Rosemary Young 3 , Tom Caradoc-Davies 3 , Emma Wanicek 1 , Ryan Hogg 1 , Hao Deng 4 , Tuncay Alan 4 , Paul Ramsland 1 5 , Nadia Zatsepin 2 , Andrew V Martin 1
  1. School of Science, RMIT University, Melbourne, VIC, Australia
  2. Department of Physics and Astronomy, Swinburne University of Technology, Hawthorn, VIC, Australia
  3. The Australian Synchrotron, ANSTO, Clayton, VIC, Australia
  4. Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC, Australia
  5. Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia

Serial synchrotron crystallography (SSX) is used to solve structures from many thousands of indexable microcrystal diffraction patterns [1]. However, when tens to hundreds of crystals are simultaneously illuminated by the beam, the patterns are impossible to index using individual peaks. Fluctuation X-ray scattering (FXS) exploits the angular correlations between Bragg peaks within such multi-crystal “spotty powder” patterns, offering a route to recover structure factors from data that established SSX analysis pipelines fail to process [2]. FXS could thus allow denser crystal loading, easier in-tray SSX data collection, potentially close the structure-solution gap between powder diffraction and SSX and considerably extend the time resolution of mix-and-diffuse SSX.

We present the first experimental demonstration of FXS from SSX data, directly benchmarking FXS-based structural analysis against established SSX merging, using the same SSX data from thaumatin microcrystals at room temperature. This is a major milestone towards our goal of retrieving molecular structure factors from non-indexable, many-crystal diffraction data and extending SSX to nanocrystals at synchrotron sources.

Data were collected at the Australian Synchrotron MX3 beamline [3] (EIGER2 XE 16M, 13 keV) from room temperature microcrystals in MiTeGen in-situ crystallisation plates, using scripts we developed for efficient fixed-target SSX data collection.  From ~230,000 frames we identified ~73,000 hits and indexed ~66,000 crystals (90% indexing rate) using CrystFEL [4]. The merged dataset (P4₁2₁2) is near 100% complete, with very high multiplicity (~500-fold overall), CC1/2 > 0.95 to 1.5 Å  (CC1/2 = 0.5 at 1.35 Å), providing a robust SSX reference dataset against which FXS-based structure factor recovery can be validated.

We computed angular correlation functions from Bragg peaks within each pattern, including from non-indexable, many-crystal patterns, and assessed recoverable correlation signal to inform our approach to structure factor retrieval via FXS beyond SSX capabilities, which we then validated against correlations computed from simulated thaumatin SSX data. Here, we present the practical requirements for FXS analysis of serial crystallography data including peak statistics, angular sampling, and correlation convergence.

This establishes thaumatin SSX as a controlled testbed for benchmarking FXS structure factor recovery against serial crystallography, with the aim of extending the approach to challenging multi-crystal patterns where conventional indexing fails. We will also present a brief overview of the novel sample delivery devices we are developing for static and time-resolved serial crystallography in support of this goal, including a 3D-printed fixed-target holder and a microfluidic flow-cell device [5] demonstrated at MX3.

  1. 1. Pearson, A. R. & Mehrabi, P. (2020). Serial synchrotron crystallography for time-resolved structural biology. Curr. Opin. Struct. Biol. 65, 168–174.
  2. 2. Adams, P., Greaves, T. L. & Martin, A. V. (2024). Crystal structure via fluctuation scattering. IUCrJ 11(4). doi:10.1107/S2052252524003932
  3. 3. Eriksson, D., Caradoc-Davies, T., Magoulas, C., Wong, D., Harnandez Vivanco, F., Cherukuvada, H., Cain, N., Rostan, R. & Nuthalapati, S. (2021). The high performance macromolecular crystallography (MX3) beamline. Presentation to the ANSTO User Meeting, 24–26 Nov 2021. https://events01.synchrotron.org.au/event/146/contributions/4378/contribution.pdf
  4. 4. White, T. A., Kirian, R. A., Martin, A. V., Aquila, A., Nass, K., Barty, A. & Chapman, H. N. (2012). CrystFEL: a software suite for snapshot serial crystallography. J. Appl. Cryst. 45, 335–341. doi:10.1107/S0021889812002312
  5. 5. Deng, H. et al. (2025). Tunable passive chaotic mixing in droplet microfluidics for controlled nanomaterial synthesis. Phys. Fluids 37, 082055. doi:10.1063/5.0281437