Instrumentation for Time-Resolved Experiments

Conference: 2021: 71st ACA Annual Meeting
08/01/2021: 12:00 PM - 3:00 PM
Oral Session 


Time-resolved crystallography utilizes the bunch structure of the X-ray source to probe the structural dynamics over a range of time scales (femtoseconds to seconds). This requires highly specialized instrumentation that can take advantage of these unique sources, as well as purpose-build serial crystallography setups. This session will focus on the current & future instrumentation and experimental setups that are needed at synchrotrons and XFELs to do these types of experiments.


Protein synchronization methods and considerations: light activation and rapid mixing

Dynamically-resolved, or better known time-resolved (TR), structural studies of proteins require efficient methods for synchronized activation followed by delivery of the sample to the X-ray beam. Deciding on the correct method of activation determines the time-resolution achievable during the experiment. Both the biological target and question, as well as the physical nature of the sample bring constraints that have to be taken into consideration, making the design of a successful time-resolved experiment is a multi-dimensional problem.

There are several current methods and tools that can be used to synchronize protein activity both in solution and in crystals. The most common approaches use either light activation or rapid mixing of substrates/ligands. Both methods have advantages and disadvantages as well as a large number of parameters that can be tuned and optimized: no technique is one-size-fits-all. As protein function synchronization is not trivial, it is unsurprising that most time-resolved structural biology experiments reported to date have focused on naturally photoactivatable proteins, as these systems can be directly activated using short laser pulses. Nevertheless, such proteins only account for roughly 0.5% of all known proteins. I have specialized in the development of both photochemical and microfluidic tools to address these challenges and will be presenting a roadmap to help guide researchers in designing their own time-resolved experiments. I will give examples of the work currently being carried out in the new synthetic chemistry laboratory at HWI, which focuses on the development of photocaged compounds. Photocaging protecting groups mask chemical moieties that are essential for function and can be removed with short pulses of light, triggering protein activity. I will also give examples of how microfluidics can be used to deliver crystal slurries to X-ray beams and how this technology can be used for efficient and fast mixing. All these tools are versatile and can be used in experiments both at synchrotron and XFELs sources.


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View Abstract 790


Diana Monteiro, Hauptman-Woodward Med. Res. Inst. Buffalo, NY 

Low background serial crystallography experiments

Latest generation (upgraded) synchrotron sources and XFELs allow generating extremely small, high-intensity X-ray beams. In the field of serial crystallography this allows performing experiments with even smaller crystals and achieving higher resolution in the diffraction experiments. For time resolved laser pump-probe experiments this significantly relaxes the requirements on the pump laser power, as only a much smaller sample volume needs to be optically excited.

Another important parameter with strong influence on the quality and achievable resolution in a diffraction experiment, and which has not received too much attention over the last years, is background scattering. Background scattering arises from the interaction of X-rays with non-sample material, including air, sample supports, and liquids around the sample. Consequently an ideal experiment requires a naked sample to be measured in a high-vacuum environment, similar to the experimental conditions in electron microscopy or in the early days of serial crystallography with liquid jets at FEL sources running in high-vacuum.

We have developed several methods with the corresponding instrumentation to significantly reduce background scattering levels in serial crystallography experiments. For the reduction air scattering, we have developed the concept of a capillary beamstop, where the direct X-ray beam is enclosed by thin-walled tantalum capillaries shortly before and behind the sample. This reduces the free-path of the beam in air to a few millimeters. Flushing this remaining free path with helium further reduces background scattering. For sample delivery we have developed micro-perforated sample holders from single crystalline silicon allowing for efficient removal of mother liquor by blotting and which do not contribute to any background scattering. More recently and following this blotting approach we have developed a low-background tape drive system for sample delivery. Here a micro-crystal suspension is applied to micro-perforated polycarbonate tape, which transports the micro-crystal suspension to the X-ray interaction point. By attaching filter paper to the backside of the perforated tape all liquid from the suspension is efficiently removed and background scattering levels are significantly reduced.

Diffraction experiments with polychromatic X-rays performed at the BioCARS instrument at the APS and ID09 at the ESRF, France, have demonstrated the applicability of these low-scattering background diffraction methods for time-resolved serial crystallography experiments and have yielded high-quality datasets. 

View Abstract 783


Alke Meents, DESY Hamburg

Additional Author(s)

Sebastian Günther, DESY Hamburg
Patrick Reinke, DESY Hamburg
Vincent Hennicke, DESY Hamburg
Pontus Fischer, DESY Hamburg

Coffee Break

Using the ALEX nylon mesh holder for time-resolved serial crystallography: successes and drawbacks.

We successfully executed a time-resolved serial crystallography experiment using our ALEX nylon mesh holder. The holder had previously been used for static structures and is not designed explicitly with light-activated systems in mind. Though we successfully captured structural dynamics by de-caging Zinc atoms using a pump laser, there are limitations and inefficiencies in the system that have drawbacks. I shall present the technical details of the experiment and discuss the pros and cons of using a nylon mesh holder for UV pump, pink beam probe, tr-SSX at BioCARS beamline at the Advanced Photon Source. 

View Abstract 583


Darren Sherrell

Additional Author(s)

Mateusz Wilamowski, Center for Structural Genomics of Infectious Diseases
Alex Lavens, Argonne National Lab Lemont, IL 
Robert Henning Plainfield, IL 

What it Takes for a Successful Mix-and-Inject Serial Crystallography Experiment: From a Sample Preparation and Sample Delivery Perspective

Serial Femtosecond Crystallography (SFX) at X-ray Free Electron Lasers (XFELs) is used to determine room temperature, damage-free, protein structures from micron-sized crystals (Chapman et al., 2011). With its demonstrated success, attention has now turned to Mix-and-Inject Serial Crystallography (MISC), which exploits the small dimensions of the microcrystals to enable rapid mixing for diffusion initiated reactions. Based on our experience with several MISC experiments on different biological systems, we present guidelines for sample preparation and injection to optimize the success of these information-rich experiments.

Samples for MISC must be comprised of a high density (~10^12 crystals/mL) of well-diffracting microcrystals. High-throughput screens are a good starting point for identifying promising microcrystallization conditions and crystal seeding is becoming increasing popular for crystal growth optimization. In terms of size, the crystals need at least one dimension below 5 micron to facilitate rapid diffusion, and they should not exceed 10-15 micron in their largest dimension to avoid injector clogging. Other important considerations include protein packing within the crystal, the accessibility of the active site, as well as the size of solvent channels that allow for the transport of ligands into the crystal. Ideally, the sample should be pre-screened to assess ligand binding by an independent technique, such as Electron Paramagnetic Resonance (EPR) spectroscopy (Calvey et al., 2020) or any other appropriately sensitive method.

For sample delivery, Gas Dynamic Virtual Nozzles (GDVNs) are a common choice as they can rapidly introduce fresh crystals for each XFEL pulse. GDVNs produce high-speed jets by exploiting a helium sheath to thin the sample stream down before the liquid exits the nozzle aperture. A robust nozzle design that uses triaxial capillaries can successfully couple a hydrodynamic focusing mixer to a GDVN for MISC (Olmos et al., 2018; Calvey et al., 2016; Calvey et al., 2019). Our lab has developed precise protocols for fabricating versatile mixers with wide sample channels (50-100 μm) to reduce clogging, that use relatively low sample flow rates (3-12 μL/min), and have the flexibility to access timepoints ranging from 3 ms-2000 ms. These injectors are compatible with different XFEL facilities, and even support vigorous remote operations during the Covid-19 pandemic (Olmos et al., 2018; Pandey et al., 2020).

All of the above, especially the ease of use of mixing injectors in remote operations, demonstrate that MISC is now routine from a sample injection perspective. The current bottleneck for MISC is the creation of high-quality microcrystals in appropriate quantities for XFEL experiments, as well as the need for new techniques to properly characterize samples in advance of beamtimes. 

View Abstract 576


Kara Zielinski, Cornell University Ithaca, NY 

Additional Author

Lois Pollack, Cornell University Ithaca, NY 

Solid-solid phase transition in adenine riboswitch crystals driven by large conformational changes induced by ligand

A solid-solid phase transition (SSPT) occurs between distinguishable crystalline forms. SSPTs have been studied extensively in metallic alloys, inorganic salt or small organic molecular crystals, but much less so in biomacromolecular crystals. In particular, SSPTs involving large-scale molecular changes that are important to biological function are largely unexplored, yet may enhance our understanding of conformational space. Here, we report a systematic study of the ligand-induced SSPT in crystals of the adenine riboswitch aptamer RNA (riboA) using a combination of polarized video microscopy (PVM), solution atomic force microscopy (AFM), and time-resolved serial crystallography (TRX). The SSPT, driven by large conformational changes induced by ligand, transforms the crystal lattice from monoclinic (apo), to triclinic (intermediate lattice in a ligand-bound conformation), to orthorhombic (final bound conformational and lattice state). Using crystal structures of each state, we mapped out the changes to the crystal packing interfaces, which define the interplay between molecular conformation and crystal phase, which were corroborated by solution AFM. Using PVM to monitor changes in crystal birefringence, we characterized the kinetics of the SSPT in crystals of different sizes and ligand concentration. Together, these studies illustrate a practical approach for characterizing SSPT in biomacromolecular crystals involving large conformational changes, and provide useful spatiotemporal data for informing time-resolved crystallography experiments. 

View Abstract 571


Jason Stagno, Center for Structural Biology, Center for Cancer Research, National Cancer Institute Frederick, MD 

Additional Author(s)

Saminathan Ramikrishnan, Center for Structural Biology, Centre for Cancer Research, National Cancer Institute Frederick, MD 
William F. Heinz, Optical Microscopy and Analysis Laboratory, Cancer Research Technology Program, Frederick National L Frederick, MD 
Valentin Magidson, Optical Microscopy and Analysis Laboratory, Cancer Research Technology Program, Frederick National L Frederick, MD 
Xiaobing Zuo, X-ray Science Division, Argonne National Laboratory Lemont, IL 
Ping Yu, National Cancer Institute Frederick, MD 
Yun-Xing Wang, Structural Biophysics Laboratory, National Cancer Institute

Diffraction and scattering studies using high brightness Excillum MetalJet X-ray sources

X-ray scattering techniques such as SAXS, BIO-SAXS, non-ambient SAXS and GISAXS rely heavily
on the x-ray source brightness for resolution and exposure time. Single crystal and powder diffraction experiments
also need the brightness and flux to measure time-based phase transitions. Traditional solid or rotating
anode x-ray tubes are typically limited in brightness by when the e-beam power density melts the
anode. The liquid-metal-jet technology has overcome this limitation by using an anode that is already
in the molten state. With bright compact sources, time resolved studies could be achieved even in the
home laboratory. We report brightness of 6.5 x 1010 photons/(sꞏmm2ꞏmrad2ꞏline) over a spot size of 10
μm FWHM.
Over the last years, the liquid-metal-jet technology has developed from prototypes into fully
operational and stable X-ray tubes running in more than 100 labs over the world. Multiple users and
system manufacturers have been now routinely using the metal-jet anode x-ray source in high-end
diffraction and scattering set-ups. With the high brightness from the liquid-metal-jet x-ray source,
novel techniques that was only possible at synchrotron before can now also be used in the home lab.
This presentation will review the current status of the metal-jet technology specifically in terms of
stability, lifetime, flux and optics. It will furthermore refer to some recent powder diffraction and
scattering data collected by our users. 

View Abstract 476


Anasuya Adibhatla, Excillum Inc Grafton, MA 

Additional Author

Julius Hållstedt, Excillum Solna

Time-resolved x-ray scattering studies on the BioCARS beam line at the APS: the nuts and bolts of pushing the limits achievable at a synchrotron facility

Time-resolved x-ray measurements using the pump-probe technique afford time resolution down to the duration of the corresponding pump and probe pulses, and precision ultimately limited by the number of photons detected. Single x-ray pulses, or short duration bursts of pulses arriving at a high repetition rate from a synchrotron facility can be isolated by a series of properly synchronized choppers and transmitted to the sample on demand by a fast millisecond shutter. When employing a synchronized short pulse laser as a pump, the time resolution achievable is limited only by the ~100 ps duration of the x-ray pulse. To maximize flux, and therefore sensitivity, the x-ray source is not passed through a monochromator, but instead its energy spectrum is kept suitably narrow for scattering studies by employing short period undulators. Though the average flux is significantly reduced by the need to gate the x-ray transmission, the peak flux is quite high, and it has proven beneficial to rapidly translate the sample to a fresh location before the next pump-pulse pair arrives, which is readily accomplished with a home-built, high-speed xyz translation stage. A home-built field-programmable-gate-array (FPGA) was developed to properly synchronize the laser, x-ray choppers and shutters, and sample position to allow rapid data collection of time-resolved, pump-probe x-ray scattering images. The signal-to-noise ratio achieved in time-resolved studies following a temperature jump (T-jump) is limited by the magnitude of the T-jump, which depends on laser pulse energy and the volume to be heated. After acquiring a secondary K-B mirror pair to enable a tighter focus at the sample location, the volume of sample that needed to be heated decreased significantly, enabling ~ 20 ˚C T-jumps in under 10 ns, and enabling time-resolved crystallography studies with significantly smaller crystals. The broad range of scattering angles and high flux available facilitate the acquisition of scattering images from crystallized biomolecules or biomolecules in solution at high spatial and time resolution. 

View Abstract 639


Philip Anfinrud, National Institutes of Health Bethesda, MD 

Additional Author(s)

Friedrich Schotte, National Institutes of Health Bethesda, MD 
Hyun Sun Cho, National Institutes of Health Bethesda, MD 
Valentyn Stadnytskyi, National Institutes of Health Bethesda, MD 

Closing Remarks