Coherent diffractive imaging of microtubules using an X-ray laser

Abstract

X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography

“…SFX has also enabled time-resolved studies of light-sensitive proteins with unprecedented temporal resolution 55 – 57 , enabling the study of reactions initiated by ligand binding and exploiting the submicrometre crystal size for rapid reaction initiation 49 – 51 , 54 , 56 – 58 . In particular, SFX has advanced fibril studies, e.g., amyloids or microtubules, where the fibrous biomolecule assemblies may have partial or no crystallinity, approaching the regime of single-molecule imaging 59 , 60 .…”

Low-dose shift- and rotation-invariant diffraction recognition imaging

“…High-resolution imaging techniques such as cryo-EM or X-ray SPI 14 , 31 , conventionally employ a low-resolution model of the molecular structure as reference to perform cross-correlation analysis for alignment of the experimental images into a high-resolution image of the structure. Currently, the EMC method (E for expansion, M for maximization and C for compression), proposed by Loh and Elser, is applied for analysis of SPI data to reconstruct a particle’s three-dimensional (3D) diffraction intensity from many photon shot-noise limited two-dimensional measurements 32 .…”

Holography and Coherent Diffraction Imaging with Low-(30–250 eV) and High-(80–300 keV) Energy Electrons: History, Principles, and Recent Trends

“…In summary, although CDI offers the theoretical possibility of recording high-resolution information, radiation damage limits the detected cpp in the diffraction pattern of a single molecule in practice, making it unsuitable to apply an iterative phase retrieval reconstruction routine to these diffraction patterns. To achieve a sufficiently strong signal for structure retrieval, other strategies such as averaging over thousands of diffraction patterns must be applied [130].…”