Dr Clyde Smith has over 30 years’ experience in the determination of small molecule and protein structures using X-ray crystallography. Dr Smith gained his PhD in Protein Crystallography with Ted Baker at Massey University in 1993, and then undertook a two-year NIH-funded postdoctoral fellowship at the University of Wisconsin with Ivan Rayment. He returned to New Zealand as a FRST postdoctoral fellow, and in 1997 he was appointed as a Lecturer in Biochemistry in the School of Biological Sciences at the University of Auckland. In late 2003 Dr Smith moved to the US to take up a Staff Scientist position in the Chemistry Department at Stanford University, working at the Stanford Synchrotron Radiation Lightsource (SSRL). He is currently a Senior Staff Scientist at SSRL. His scientific research in the field of structural biology includes projects in antibiotic resistance, folate metabolism, vitamin B12 chemistry, plant dirigent proteins, soil viruses, serial crystallography and time-resolved crystallography.

SSRL serial microcrystallography: The automated instrumentation employed at the highly productive micro-focus beamlines, SSRL 12-1 and 12-2, include automated instrumentation and remote access. Their capabilities are also being expanded to enable time-resolved experiments to study metalloenzyme structure and protein dynamics using novel photo-triggering and chemical mixing methods. These include serial diffraction studies using mixing injectors, microfluidics, flow cells, rapid freeze-quenching, and the incorporation of photo-caged reactants within crystals.

Dr. Junko Yano is a Senior Scientist at the Lawrence Berkeley National Laboratory, in the Molecular Biophysics and Integrated Bioimaging Division and Liquid Sunlight Alliance.  She is a leading scientist in the field of photosynthetic systems and metalloenzymes.  Over the last decade, Dr. Yano led numerous pioneering experiments using pump probe serial femtosecond crystallography (SFX) and simultaneous X-ray emission spectroscopy at X-ray free-electron lasers (XFELs).  The group is also active in developing sample delivery and reaction triggering methods that include a drop-on-tape method for efficient crystallography data collection at XFELs.  The most noticeable work by the group is their study on Photosystem II (PSII), a multi-subunit membrane protein, that catalyzes the light-driven water oxidation reaction in nature.  Understanding the water oxidation reaction catalyzed by the Mn4CaO5 cluster in PSII has been one major research focus over many years.  The group is investigating how the protein environment works together with metal catalytic center to enable multielectron/proton reaction, by capturing its structural and chemical changes during the water oxidation reaction.  In the recent study, the group showed a sequence of events that are related to substrate insertion and proton release mechanism during the catalysis by crystallography.  She has also applied the simultaneous X-ray crystallography/X-ray spectroscopy technique to study various metalloenzymes, by triggering reactions in situ at room temperature using XFELs.  These enzymes include Isopenicillin N synthase, Methane Monooxygenase, Methyl-Coenzyme M. Reductase, and others.  

Some recent relevant papers include: 

• Structural dynamics within the water and proton channels of Photosystem II Biophysical Journal 2022

• Effects of x-ray free-electron laser pulse intensity on the Mn K β 1, 3 x-ray emission spectrum in photosystem II—A case study for metalloprotein crystals and solutions Structural Dynamics 2021

• Structural dynamics in the water and proton channels of photosystem II during the S2 to S3 transition Nature Comm. 2021

• An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography Nature Comm. 2021

• Using X-ray free-electron lasers for spectroscopy of molecular catalysts and metalloenzymes Nature Rev. Phys. 2021

• Untangling the sequence of events during the S2→ S3 transition in photosystem II and implications for the water oxidation mechanism PNAS 2020

Dr. J. Nathan Hohman is an Assistant Professor of Chemistry at the University of Connecticut. His academic journey began at Penn State where he earned his doctorate in Chemistry. This led to post-doctoral work at Stanford University's Department of Materials Science and Engineering and a staff scientist position at the Molecular Foundry at Lawrence Berkeley National Laboratory. He started his independent research there, focusing on creating novel inorganic materials with unique optical properties. In the Hohman lab group, he leads a team in controlling and designing inorganic coordination polymers, with the goal of understanding their structure and function relationships. The development of small-molecule serial femtosecond crystallography at X-ray free electron laser (XFEL) beamline facilities to support this research is a key focus area. Dr. Hohman is the lead PI on the Integrated Computational and Data Infrastructure (ICDI) program, a collaboration between the University of Connecticut, Lawrence Berkeley National Laboratory, and Massachusetts Institute of Technology.

The ready availability of single crystal X-ray diffraction has led to a wealth of new materials characterized each year. However, known material structures are biased towards those that are easily crystallized. Hybrid materials in particular trend towards crystals too small or otherwise pathological to be used for traditional characterization techniques. These “dark” materials that are too difficult to characterize make up a large portion of hypothetical hybrid materials. Here, we used a new technique of serial femtosecond chemical crystallography (SFCX) that uses the high brightness of an X-ray free-electron laser to acquire diffraction from crystals in the 1-5 micron range. Graph theory is used to index those snapshots, enabling the determination of crystal structure. We used this technique to explore the ligand environment with variables of steric hindrance, functional group, and intermolecular forces, each addressed by selecting different ligand shapes and configurations. We find dramatic differences in the connectivity, topology, and dimensionality of the resulting silver organothiolates. Of note is the nature of the Ag-Ag interactions, which appear to be an important component of the fine structure of the silver systems. The Ag-Ag networks are found to rearrange as a function of the supramolecular ordering of each example system

Alessandra Henkel received her bachelor’s and master’s degree in Molecular Life Science at the University of Lübeck, Germany. She was introduced to macromolecular crystallography by Prof. Dr. Lars Redecke during her bachelor thesis on in cellulo crystallization and pursued working in the field of serial crystallography during her master thesis at beamline P11 under the supervision of Johanna Hakanpää, PhD. During this time, she developed the idea of JINXED. In a subsequent internship, supervised by Dr. Dominik Oberthür, at the Center for Free-Electron Laser Science (group Prof. Henry Chapman), she started working on the development of sample delivery methods for serial crystallography, which continued into her current PhD project Reliable and Efficient Multi-dimensional Serial Crystallography. 

Macromolecular crystallography is a well-established method in structural biology and after focusing on static structures, the method is now developing towards the investigation of structural dynamics, e.g. by looking at protein-ligand or enzyme-substrate interactions. In time-resolved serial crystallography and room-temperature data collection, the reaction is triggered within the crystals – either optically or chemically. For the latter, the use of micron-sized crystals is necessary to ensure short diffusion times and quick saturation within each crystal. However, certain crystal morphologies e.g. small solvent channels can prevent sufficient ligand diffusion. Presented here is a method combining protein crystallization and data collection in a novel one-step-process to overcome the aforementioned challenges. We successfully performed corresponding experiments as a proof-of-principle using hen egg-white lysozyme with crystallization times of a few seconds. This method called JINXED (Just in time crystallization for easy structure determination) promises to result in high-quality data due to the avoidance of crystal handling and could enable time-resolved experiments regardless of crystal morphology by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches. In combination with upcoming automated data processing, this method may offer the possibility to combine high-throughput ligand screenings and detailed dynamical investigations with a high level of automation.

Dr Eleanor Campbell is a beamline scientist for the MX beamlines at the Australian Synchrotron. She provides support to beamline users and assists in the maintenance and development of the MX1 and MX2 beamlines.  

Her research interests are predominantly based around molecular evolution; how and why proteins evolve in the ways that they do, both in nature and in laboratory experiments. Before joining the MX team at the Australian Synchrotron, Eleanor held a postdoctoral position at the University of Cambridge, where she provided support to colleagues in need of structural biology expertise. In addition to her research at the University of Cambridge, Eleanor acted as the Network Manager for an EU H2020 grant, coordinating 16 PhD students across 10 institutes in collaborative research projects.

Eleanor is passionate about communicating science to broad audiences, and has previously worked as a writer for CSIRO’s children’s magazine, The Helix, and as a radio and podcast host for a number of science shows. 

MX3

The High-Performance Macromolecular Crystallography beamline at the Australian Synchrotron (MX3) will be capable of providing high-flux, microfocus X-rays for small and weakly diffracting protein crystals. The beamline will provide three modes of operation: goniometer, serial crystallography (including fixed target and injector sample delivery) and in-tray screening. MX3 will have a rapidly adjustable beam size and specialise in high flux, high-speed data collection on microcrystals with a high degree of automation for crystal location and data collection. 

Dr Nadia Zatsepin is an ARC Future Fellow working in the Department of Mathematical and Physical Sciences at La Trobe University. She works on the development and application of serial femtosecond crystallography (SFX) with XFELs and its adaptation to synchrotron sources with the goal of making molecular movies. Previously she led SFX data analysis in the NSF BioXFEL Science and Technology Center while working at Arizona State University, and was part of the ARC Centre of Excellence in Advanced Molecular Imaging. More info here: Zatsepin lab site.