Fast high-pressure freezing of protein crystals in their mother liquor

Abstract

High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts.

Figure 1

A tHEWL crystal in a quartz capillary submerged in 1-hexadecene (picture taken under a polarizing microscope): A, mother liquor; B, tHEWL crystal.

Figure 2

Sample preparation for HPF. Method A (front): A, aluminium lid; B, quartz capillary containing the crystal in its mother liquor; C, aluminium platelet with cavity filled with 1-hexadecene. The sample is sandwiched between the platelet and the lid under 1-hexadecene and is then transferred to the specimen holder. Method B (back): D, aluminium lid; E, Kapton foil; F, Teflon platelet containing the crystal in its crystallization buffer. Sample preparation is performed in the specimen holder, which is subsequently inserted into the HPF device.

Figure 3

Baltec HPM 010 high-pressure freezing device.

Figure 4

Diffraction image of an HPF cubic insulin crystal collected at beamline X06DA at the Swiss Light Source. The red ring indicates a resolution of 1.9 Å. An enlarged picture of the boxed area showing Bragg reflections at 1.9 Å resolution is shown at the bottom right.

Figure 5

Diffraction image of an HPF crystal of PSII collected at beamline I24 at Diamond Light Source. The red ring indicates a resolution of 4.5 Å. An enlarged picture of the boxed area showing Bragg reflections at 4.5 Å resolution is shown at the bottom right.