A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering

Abstract

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller.

Protein Micro-Crystallography: Nanotechnology Challenges Ahead

“…Indeed, scattering from a 10 μm single-crystalline silicon membrane [37] is very low for a transmission of ~97 % at λ~0.1 nm wavelength. The [41].…”

“…An example for this is provided by a silicon chip with a holepattern etched into a 10 μm membrane by photolithography techniques ( Figure 6A) [37]. Evaporating a droplet containing a slush of crystallites on the chip allows immobilizing individual crystallites in the down to 1 μm diameter holes by capillary action ( Figure 6B).…”

TakeTwo: an indexing algorithm suited to still images with known crystal parameters

“…The diffraction patterns used for CPV17 had previously been used for structure determination Ginn, Messerschmidt et al, 2015). Data were collected at the XPP endstation at the Linac Coherent Light Source (LCLS) under proposal LH90 from BEV crystals on a silicon chip (Roedig et al, 2015; cubic space group F23, unit-cell dimension 437 Å ). Reflections were observed to approximately 2.0 Å resolution and the minimum separation of individual reflections was three pixels.…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…These issues are then further compounded by the need to deliver those samples efficiently to the X-ray beam. 5,[7][8][9][10][11]18,[37][38][39][40][41][42] Microfluidic and microscale technologies have played a critical role in facilitating both protein crystallization and structure determination, with the breadth and variety of reported solutions demonstrating the challenging nature of the field. In this review, we discuss the utility of microfluidic technologies for addressing the challenges of crystal growth and sample manipulation for applications in serial crystallography and time-resolved structure determination.…”