Cryo Data Collection

What is Cryo data collection?

Cooling the crystal during x-ray data collection to temperatures below the melting point of the mother liquor or solution that it is in equilibrium with, typically to between -150 and -180 C.

Why Cryo data collection?

reduce radiation-induced loss of order and thus intensity of diffraction (dose/time-dependent as well as resolution-dependent)
reduce thermally-induced decay (heat stress, usually only an issue at synchrotrons)
trap short-lived reaction intermediates
ease of mounting & storage
lower temperature factors

Without cryo, in most cases partial datasets from many separate crystals need to be merged to obtain a complete high-resolution dataset.

The cause of radiation-induced crystal decay

The cause of radiation-induced crystal decay are photochemically produced free radicals which react with the protein randomly, breaking down the order of the lattice. Disruption of order in the crystal results in dose-dependent decay of the intensities of individual Bragg reflections.

photochemical production of free radicals is largely temperature-independent
diffusion-dependent propagation of free radicals and their reaction rates with macromolecules are greatly reduced at cryo temperatures

History

initially developed for small molecule x-ray crystallography (40s-60s)
first successful application for a protein crystal by Haas & Rossmann (1970)
further developed independently by

Greg Petsko (cryoprotectants, 1975)
Hakon Hope (oil, 1988)

further developed in the 90s with increasing availability of synchrotron x-ray sources

Special considerations

Avoid ice formation. Gradual lowering of the temperature causes formation of ice crystals around or within the protein crystal lattice which degrades the diffraction from the protein lattice by

contributing to the diffraction pattern (ice rings)
shearing the crystal lattice (decreased order of protein lattice)

The solution is to flash-cool the protein crystal by plunging it into liquid nitrogen. With rapid cooling and the aid of cryoprotectants, only amorphous (or vitreous) ice is formed. Useful cryoprotectants are often alcohols, sugars, amino acids or sulfoxides, which

slow the nucleation of ice
raise the viscosity of the solution (and thus raise its glass transition temperature)
break the propagation of ice formation in the mother liquor

Cryoprotectants are usually added in amounts ranging from as low as 2% to over 30% by volume.

Common problems

deleterious effects of cryoprotectant on protein crystal
increased mosaic spread (in rare cases a decrease)
change in unit cell constants (usually shrinking by several %)

Other parameters that affect the cooling process:

size and shape of the crystal
mechanical stability of the crystal

Other benefits

reduced thermal vibrations (lower B factors)
enhanced signal-to-noise ratio (less background and absorption)
reduced conformational disorder (freezing out of few, preferred conformations)
in many cases, higher limiting resolution

Practical Aspects

Mounting methods

loop (material, size, thickness)
material, length and thickness of pin holding the loop
storage in cryo vials (shipping)

Cooling methods

Straight into gaseous stream
plunge into liquid nitrogen (faster cooling rate), then transfer into stream
plunge into liquid propane (higher heat conductance, less bubble formation)

Minimize icing during data collection by reducing stream turbulence

proper distance and centering of cryo nozzle
co-axial cryo stream
inner cold stream surrounded by dry, warmer outer stream
heated conical deflector on goniometer head
low ambient humidity (less than 40% r.h.)
control of drafts that could deflect cryo stream
use thin pin to minimize stream turbulence

References & weblinks

Practical Cryocrystallography by D.W. Rodgers, Meth. Enz. 276, 183-203 (1997).
Structural changes in a cryo-cooled protein crystal owing to radiation damage (W. Burmeister)
Cryo-Notes from SSRL Workshop October 1995 (Hakon Hope)
Flash-Cooling: A Practical Guide
Cryogenic X-ray data collection
High Resolution Cryo-Crystallography of Biological Macromolecules
LMB step-by-step cryo-crystallography tutorial guide
Liquid Nitrogen Fun!

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