Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.7 Å inside cells

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and in situ. In addition to aligning individual particles, accurate registration of sample motion and 3D deformation during exposures is crucial for achieving high resolution. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and improves the results of structure determination. M provides a unified optimization framework for both in vitro frame series and in situ tomographic tilt series data. We show that tilt series data can provide the same resolution as frame series, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M improves upon previous methods, and resolves a 70S ribosome bound to an antibiotic inside bacterial cells at a nominal resolution of 3.7 Å. Thus, computational tools are now available to resolve structures from tomographic in situ cryo-EM data at residue level.

Dynamic super-resolution structured illumination imaging in the living brain

Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

KB scanning of X-ray beam for Laue microdiffraction on accelero-phobic samples: application toin situmechanically loaded nanowires

A mapping technique has been developed where a sub-micrometer focused polychromatic X-ray beam is scanned across a stationary sample instead of scanning the sample in front of the X-ray microbeam. This method is applied to a gold nanowire during its mechanical loading using the tip of an atomic force microscope. During the loading process, such a sample is `accelero-phobic',i.e.the sample scanning stages must not to be moved to avoid parasitic additional load. Without beam scanning, only one single position within the sample can be probed during the test. The probed material point may even change because of drifts or movements induced by the test itself. The new scanning approach facilitates thein situmapping of the entire wire giving access to the evolution of the wire shape as well as to the boundary conditions. This novel scanning technique opens promising perspectives for studies where sample motion is forbidden because of the sample environment.

Utilizing broadband X-rays in a Bragg coherent X-ray diffraction imaging experiment

A method is presented to simplify Bragg coherent X-ray diffraction imaging studies of complex heterogeneous crystalline materials with a two-stage screening/imaging process that utilizes polychromatic and monochromatic coherent X-rays and is compatible within situsample environments. Coherent white-beam diffraction is used to identify an individual crystal particle or grain that displays desired properties within a larger population. A three-dimensional reciprocal-space map suitable for diffraction imaging is then measured for the Bragg peak of interest using a monochromatic beam energy scan that requires no sample motion, thus simplifyingin situchamber design. This approach was demonstrated with Au nanoparticles and will enable, for example, individual grains in a polycrystalline material of specific orientation to be selected, then imaged in three dimensions while under load.

Anisotropic Correction of Beam-induced Motion for Improved Single-particle Electron Cryo-microscopy

Correction of electron beam-induced sample motion is one of the major factors contributing to the recent resolution breakthroughs in cryo-electron microscopy. Improving the accuracy and efficiency of motion correction can lead to further resolution improvement. Based on observations that the electron beam induces doming of the thin vitreous ice layer, we developed an algorithm to correct anisotropic image motion at the single pixel level across the whole frame, suitable for both single particle and tomographic images. Iterative, patch-based motion detection is combined with spatial and temporal constraints and dose weighting. The multi-GPU accelerated program, MotionCor2, is sufficiently fast to keep up with automated data collection. The result is an exceptionally robust strategy that can work on a wide range of data sets, including those very close to focus or with very short integration times, obviating the need for particle polishing. Application significantly improves Thon ring quality and 3D reconstruction resolution.