Coherent X-ray diffraction imaging of single particles: background impact on 3D reconstruction

Area of Science:

• Structural biology
• X-ray science
• Biophysics

Background:

• Coherent diffractive imaging (CDI) with X-ray free-electron lasers (XFELs) offers potential for room-temperature macromolecular structural studies.
• Studying femtosecond structural dynamics is a key application, but weak protein diffraction is challenged by background scattering.

Purpose of the Study:

• To evaluate the impact of background noise on the CDI analysis pipeline for macromolecular structures.
• To determine the signal-to-background ratio (SBR) requirements for successful 3D reconstructions.

Main Methods:

• Combined experimental background data from the European XFEL with simulated diffraction patterns.
• Applied standard reconstruction procedures, including orientation recovery (expand, maximize, and compress algorithm) and 3D phase retrieval.

Main Results:

• Background scattering negatively affected the resolution of reconstructed density maps.
• Successful reconstructions were achieved with an SBR of 0.6 or higher in the relevant resolution shell.
• The required SBR increases with higher resolution.

Conclusions:

• CDI at XFELs is viable with current background levels, provided adequate SBR.
• Achieving high-resolution structural information necessitates optimizing experimental conditions to minimize background noise.
• This study provides benchmarks for current XFEL capabilities and targets for background reduction.