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.
Highlights
Bound tubulin dimer can depolymerize from the microtubule, giving rise to what is termed dynamic instability[5] during which microtubules rapidly switch between cycles of growth and shrinkage within the cell
Microtubule suspensions were injected in vacuum across a highly focused (~200 nm focal diameter) X-ray free electron lasers (XFELs) beam using a gas dynamic virtual nozzle[34,35] at the Coherent Xray Imaging (CXI) experimental station[41] of the Linac Coherent Light Source (LCLS)[42] at the SLAC National Accelerator Laboratory
This extends earlier reports of X-ray diffractive imaging of other filamentous systems[31,32,33] by using imaging sorting techniques pioneered for electron microscopy to sort and average images[36], and by applying a simple iterative phase retrieval to recover 2D projection images of microtubules. This approach allowed data to 2 nm resolution (Fig. 2c, d) to be incorporated into the reconstruction from which structural details became visible with a characteristic length scale of 4 nm (Fig. 5d). In this manner the amplification of the diffraction signal due to the presence of 1D translational symmetry has facilitated a significant advance in resolution over earlier coherent diffractive imaging studies of virus particles using XFEL radiation[24,26] that achieved a resolution of 32 nm in projection[24] and 125 nm[26] and 28 nm[27] after 3D reconstruction, or projection images of live cyanobacteria[50] and carboxysome[28] that were recovered to a resolution of 75 nm and 18 nm respectively
Summary
Bound tubulin dimer can depolymerize from the microtubule, giving rise to what is termed dynamic instability[5] during which microtubules rapidly switch between cycles of growth and shrinkage within the cell. Microjet diameter and the XFEL beam focus, ~20 microtubules were sampled within the exposed volume for every XFEL exposure We sorted these images into class averages using software adapted from single-particle cryo-electron microscopy applications[36,37]. Data were merged to recover a single 2D diffraction pattern extending to 2 nm resolution from which we made a 2D projection image reconstruction using iterative phase retrieval assuming that the density outside of the microtubule was zero[24,38,39] This analysis recovered the characteristic diameter of the microtubule distributions and resolved 4 nm sub-structure corresponding to the individual tubulin monomers that was not included in the initial phases. Future advances in XFEL intensity and focus, as well as improving sample handling and injection procedures, may allow dynamical processes to be imaged at room temperature to high resolution
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
