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A Beginner’s Guide to Understanding Radiation Damage in Macromolecular Crystallography

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Radiation damage is a significant challenge in macromolecular crystallography, especially with the advent of high-brightness synchrotron sources. This beginner’s guide explores the fundamental aspects of radiation damage, its impact on structural determination, and strategies to mitigate its effects, enabling more accurate and reliable structural models.

The Nature of Radiation Damage

Radiation damage in crystals arises from the interaction of X-rays with the sample. When X-rays interact with matter, they cause ionization and excitation of atoms. In the context of macromolecular crystals, this energy absorption leads to the formation of free radicals and other reactive species. These highly reactive molecules then attack the macromolecules within the crystal, causing various forms of damage.

Types of Radiation Damage

There are two broad categories of radiation damage:

  1. Specific Damage: This type of damage involves the direct interaction of X-rays with specific chemical bonds, leading to their breakage. For example, disulfide bonds are particularly susceptible to specific damage.

  2. Non-Specific Damage: This involves the indirect effects of radiation, mediated by secondary species such as free radicals. This type of damage can affect a wide range of chemical bonds and amino acid residues.

Schematic of an X-ray diffraction experimental setup for macromolecular crystallography, illustrating the interaction of X-rays with the crystal and subsequent diffraction pattern.

Consequences of Radiation Damage

The consequences of radiation damage are diverse and can significantly impact the quality of crystallographic data. Some common effects include:

  • Loss of Diffraction Resolution: As the crystal is exposed to radiation, the overall order within the crystal lattice decreases. This manifests as a reduction in the intensity of high-resolution reflections, effectively limiting the resolution of the data.
  • Changes in Unit Cell Parameters: Radiation damage can cause subtle changes in the unit cell dimensions of the crystal, affecting data processing and merging.
  • Increased Mosaic Spread: The mosaic spread, which reflects the degree of disorder within the crystal, tends to increase with radiation exposure.
  • Introduction of Artifacts: Changes in electron density, particularly around sensitive residues such as cysteines, can introduce artifacts into the structural model.

Factors Influencing Radiation Damage

Several factors influence the rate and extent of radiation damage in macromolecular crystals.

Wavelength

The wavelength of the X-rays used for diffraction plays a crucial role in radiation damage. Shorter wavelengths (higher energy) tend to cause more ionization events, leading to increased radical production and, consequently, more damage.

Temperature

Crystals are often cryo-cooled to reduce radiation damage, but very low temperature can lead to other problems.

Chemical Composition

The chemical composition of the crystal environment also plays a role. The presence of reducing agents, such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), can scavenge free radicals, mitigating the effects of radiation damage.

Strategies for Mitigating Radiation Damage

Given the pervasive nature of radiation damage, various strategies have been developed to minimize its impact on structural determination.

Cryo-cooling

Cooling crystals to cryogenic temperatures (typically around 100 K) dramatically reduces the mobility of free radicals, slowing down the chemical reactions that lead to damage. This is the most widely used method for mitigating radiation damage.

Illustration of a cryocooled protein crystal, showing the reduction in atomic mobility at low temperatures, which helps to minimize radiation damage.

Crystal Treatment

Soaking crystals in solutions containing cryoprotectants, such as glycerol or polyethylene glycol (PEG), can improve their resistance to radiation damage. In addition, adding radical scavengers to the cryoprotectant solution can further reduce damage.

Data Collection Strategies

Optimizing data collection strategies can also help minimize radiation damage. These strategies include:

  • Multi-Crystal Method: Collecting data from multiple crystals and merging the data sets allows for lower doses per crystal, reducing overall damage.
  • Wedge Method: Collecting data in narrow wedges of oscillation can minimize the exposure time for each part of the crystal.

Anomalous Diffraction Techniques

Using anomalous diffraction techniques, such as single-wavelength anomalous diffraction (SAD) or multi-wavelength anomalous diffraction (MAD), can help determine structures from data collected with lower doses of radiation.

Assessing Radiation Damage

It is important to monitor the progression of radiation damage during data collection to ensure the quality of the final structural model. Several methods can be used to assess the extent of damage.

Monitoring Diffraction Quality

The most straightforward method is to monitor the diffraction quality as data collection proceeds. This can be done by tracking the resolution limit, the intensity of high-resolution reflections, and the overall mosaicity of the crystal.

R-factor Analysis

The R-factor (residual factor) is a measure of the agreement between the observed and calculated diffraction intensities. An increase in the R-factor can indicate the onset of radiation damage.

Isotropic B-factor analysis

The isotropic B-factor is a parameter that describes the average displacement of atoms from their mean positions. An increase in the overall B-factor can be indicative of radiation damage.

Occupancy Refinement

Monitoring changes in the occupancy of specific residues, such as cysteines, can provide insights into the progression of specific damage.

Advanced Techniques

More advanced methods, such as serial femtosecond crystallography (SFX), are being developed to outrun radiation damage.

Serial Femtosecond Crystallography (SFX)

In SFX, a stream of nanocrystals is hit with extremely short, intense X-ray pulses from an X-ray free-electron laser (XFEL). Diffraction data is collected before the crystal is destroyed. SFX allows structure determination free from radiation damage at room temperature, offering new insights into protein dynamics and function.

Conclusion

Radiation damage remains a challenge in macromolecular crystallography, but its effects can be mitigated through careful experimental design and data collection strategies. By understanding the nature of radiation damage and employing appropriate techniques, it is possible to obtain high-quality structural data, leading to more accurate and reliable structural models. This beginner’s guide provides a foundation for understanding and addressing radiation damage, enabling researchers to push the boundaries of structural biology.

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