Abstract
The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.
Highlights
The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data
Cytoplasmic polyhedrosis viruses (CPVs) are protected by a robust case, each a tiny crystal of viral polyhedrin, whose volume varies from 10 À 3 to 103 mm[3] depending on the CPV type
We are able to determine the structure by molecular replacement, which in our hands fails on a lower-resolution 100 K data set of CPV type 17 (CPV17) collected at a synchrotron
Summary
The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. We use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We apply more sophisticated methods of SFX data analysis and show that the quality of the measurements can be improved, allowing us to obtain one of the highest-resolution structures published to date (1.75 Å) from an XFEL, using 5,787 crystals of CPV type 17 (CPV17), fewer than previously required for high-resolution analysis[8,9,10,11]. We here demonstrate the power of such a method tailored to and applied to real XFEL data, revealing the promise of such improved models to allow XFELs to tackle more challenging biological samples
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