Microfabricated mounts for high-throughput macromolecular cryocrystallography

Abstract

A new approach is described for mounting microcrystals of biological macromolecules for cryocrystallography. The sample mounts are prepared by patterning thin polyimide films by standard microfabrication techniques. The patterned structures contain a small hole for the crystal connected to a larger hole via a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. These polyimide structures are wrapped around small metal rods. The resulting curvature increases their rigidity and allows a convenient scoop‐like action in retrieving crystals. The polyimide contributes minimally to X‐ray background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity. The new mounts are fully compatible with existing automated sample‐handling hardware for cryocrystallography. Their potential advantages include completely reproducible sample hole sizes to below 10 µm; accurate and reproducible sample positioning and good sample‐to‐mount contrast, simplifying alignment; more convenient manipulation of small crystals; easier removal of excess liquid and reduced background scatter; reduced thermal mass and more rapid flash‐cooling; and easy design customization and mass production. They are especially well suited to data collection from the smaller crystals produced in high‐throughput crystallization trials, and are suitable for automated crystal retrieval. They should be more generally useful for X‐ray data collection from small organic and inorganic crystals of all types.

A study on new approaches in coherent x-ray microscopy of biological specimens

“…25 The sample support and mounting, on the other hand, is based on a design [144] that has been devised for cryo protein crystallography and profits from the well-developed technical tools and infrastructure in this field [51]. For the actual sample support a thin microfabricated polyimide foil 26 was used, mounted onto a stainless steel pin with a diameter of 0.64 mm (MiTeGen, USA), see Figs.…”

Operator-assisted harvesting of protein crystals using a universal micromanipulation robot

“…With some practice, an operator was able to capture these crystals successfully via the robot's keyboard-driven user interface. Tests were performed using both standard nylon loops provided by Hampton Research and etched Kapton loops manufactured by MiTeGen (Thorne et al, 2003). While both types of loops worked, the Kapton loops were easier to manipulate relative to the crystal and allowed the capture sequence to be performed more quickly.…”

“…Hampton Research CrystalCap pins containing the cryoprotected robotically harvested crystals in MiteGen Kapton loops (Thorne et al, 2003) were directly mounted out of liquidnitrogen storage pucks using cryo-tongs. Diffraction data of the orthorhombic P2 1 2 1 2 1 crystals were collected in anomalous data collection strategy at 100 K with a Platinum 135 CCD detector using Cu K radiation from a Bruker MICROSTAR-H rotating-anode generator operating at 2.7 kW.…”

“…The UMR arm is equipped with a versatile tool exchanger. Tape cutters, harvesting pins with MiTeGen MicroLoops (Thorne et al, 2003), cryoliquid exchange and custom tools provide additional operational flexibility. The current user interface is a simple game-style keyboard layout allowing intuitive manual control.…”

In vacuoX-ray data collection from graphene-wrapped protein crystals

“…Major sources of X-ray background include the solvent surrounding the crystal and the crystal mount or loop. Crystal mounts have been adapted to include a drainage channel to help remove any excess liquor around the crystal, whilst being constructed from polyimide to contribute minimally to background scatter (Thorne et al, 2003). The air paths before and after the sample, up until the X-ray beamstop, or the cryo-gas stream, also contribute significantly to the background scatter.…”