Supports for serial protein crystallography

In close collaboration with the macromolecular crystallography group at PSI and the Institute of Polymer Nanotechnology at PSI/FHNW, we are developing solid supports for serial protein crystallography at the SLS and at SwissFEL [1-3].

Serial femtosecond crystallography (SFX) is a powerful new method for protein structure determination at X-ray free electron lasers (XFELs) and synchrotron X-ray sources [4, 5]. It is based on collecting diffraction patterns from large numbers of small protein crystals and allows solving protein structures directly from protein microcrystals, which are too small for standard X-ray techniques. Furthermore, the femtosecond short duration of XFEL pulses allows for dynamic studies of fast structural changes in proteins using pump-probe techniques [6].

To deliver the protein microcrystals at high frequency to the probing X-ray beam, novel protein crystal handling methods such as liquid jet and viscous media injection technologies, as well as the so-called fixed target technology have been developed [7, 8]. In the latter approach, the crystalline sample is deposited on a thin film support, which is mounted on a scanning stage and scanned through the beam, thus sequentially probing the individual microcrystals with the tightly focused x-rays.

The supports are usually fine grid structures or microporous membranes, traditionally produced using silicon/silicon nitride technology, onto which the protein crystal suspension is deposited and separated from the mother liquid through their sieve-function.

We are focusing on the development and fabrication of polymer-based devices, thus taking advantage of the low X-ray absorption and scattering background of polymer materials, absence of X-ray diffraction if using amorphous polymers, the high design flexibility and the potential mass-fabrication at low cost.

MISP-Chip
An overview of the MISP chips. (a) The two current versions of the MISP chip with transparent COP and black COC membrane. The black scale bar shown on the frame of the transparent COP MISP chip represents 10 mm. (b) The parameters used to define the chips. (c) Magnified fiducials (red box), cavities (green box), cavity profiles (green) and label.

MISP-Chips (Microstructured Polymer Chips)

  • developed for RT-serial protein crystallography at SwissFEL/CristallinaMX
  • apperture-alignment of crystals 
  • Small – 6,000 apertures
  • Large – 29,000 apertures
  • Aperture pitch = 120 µm
  • Typically sealed in two layers of Mylar film
    standard 6 µm, but thinner films are possible.
  • Suitable for SFX and SFX pump-probe
Polymer support for SFX
A 3 µm thick perforated COC film is suspended in a 5 x 5mm sized polymer frame and fixed on a pin as used for protein crystallography. The SEM images on the right show 3 µm sized perforations in the membrane generated with nanoimprint lithography.
microcrystals and diffraction image
Lysozyme microcrystals deposited on a perforated polymer membrane of a SwissMX support after blotting of the mother liquid and flash-cooling. Right: Diffraction pattern from one of the crystals recorded at SwissFEL.

SwissMX supports

  • developed for serial protein crystallography at XFELs and synchrotrons
  • minimized background
  • efficient blotting
  • solutions for cryo and RT measurements
  • optimized for use at the SwissMX instrument at SwissFEL
  • adaptable to other formats
  • fabrication based on planar technology and additive manufacturing

For more information and test samples
please contact Celestino Padeste.

References:

[1] M. Carrillo et a., Micro-structured polymer fixed targets for serial crystallography at synchrotrons and XFELs. IUCrJ (2023) 10, 678-693 doi: 10.1107/S2052252523007595.

[2] A. Karpik, I. Martiel, P.M. Kristiansen, C. Padeste, Fabrication of ultrathin suspended polymer membranes as supports for serial protein crystallography, Micro and Nano Engineering. 2020; 7: 100053 (6 pp.).

[3] I. Martiel et al., Versatile microporous polymer-based supports for serial macromolecular crystallography, Acta Cryst. (2021). D77, 1153–1167, doi: 10.1107/S2059798321007324.

[4] S. Boutet et. al., High-resolution protein structure determination by serial femtosecond crystallography. Science 337 (2012) Vol. 6092, pp. 362-364.

[5] T. Weinert et al., Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons, Nat. Commun. 8, 542 (2017).

[6]  V. Paneels et al., Time-resolved structural studies with serial crystallography: A new light on retinal proteins. Structural Dynamics, 2, 041718 (2015)

[7 I. Martiel, H.M. Müller-Werkmeister & A.E. Cohen, Strategies for sample delivery for femtosecond crystallography. Acta Crystallogr. Sect. D. 75, 160–177 (2019).

[8]  M.S. Hunter et al., Fixed-target protein serial microcystallography with an x-ray free electron laser, Sci. Rep. 4, 6026 (2014).