Dose, exposure time and resolution in serial X-ray crystallography.

Area of Science:

• Structural Biology
• Biophysics
• X-ray Imaging

Background:

• X-ray diffraction microscopy resolution is limited by sample radiation damage.
• High doses required for resolution cause irreversible damage to biological samples.

Purpose of the Study:

• To present a serial crystallography method to overcome radiation damage limitations.
• To determine the X-ray fluence required for high-resolution imaging.
• To assess the feasibility of achieving sub-nanometer resolution with current and future X-ray sources.

Main Methods:

• Serial crystallography using a continuous X-ray beam and hydrated macromolecules in a single-file train.
• Laser alignment for orienting diffracting molecules.
• Analytical modeling, explicit simulation, and transfer function analysis to evaluate X-ray fluence requirements.
• Iterative phase problem solution and electron density map reconstruction.

Main Results:

• Resolution is limited by X-ray flux and molecular alignment, not solely by radiation damage.
• An inverse fourth-power dependence of exposure time on resolution was confirmed.
• Sub-nanometer resolution requires multiple protein beams on current synchrotrons, but is feasible on fourth-generation sources.
• 7 Å resolution of secondary protein structure is achievable with short exposures on future sources.

Conclusions:

• Serial crystallography offers a viable path to high-resolution structural determination of macromolecules.
• Advancements in X-ray sources (fourth-generation) are crucial for routine high-resolution imaging.
• The method has significant implications for coherent X-ray imaging techniques.