Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

Protein orientation in time-dependent electric fields: orientation before destruction.

Proteins often have nonzero electric dipole moments, making them interact with external electric fields and offering a means for controlling their orientation. One application that is known to benefit from orientation control is single-particle imaging with x-ray free-electron lasers, in which diffraction is recorded from proteins in the gas phase to determine their structures. To this point, theoretical investigations into this phenomenon have assumed that the field experienced by the proteins is constant or a perfect step function, whereas any real-world pulse will be smooth. Here, we explore the possibility of orienting gas-phase proteins using time-dependent electric fields. We performed ab initio simulations to estimate the field strength required to break protein bonds, with 45 V/nm as a breaking point value. We then simulated ubiquitin in time-dependent electric fields using classical molecular dynamics. The minimal field strength required for orientation within 10 ns was on the order of 0.5 V/nm. Although high fields can be destructive for the structure, the structures in our simulations were preserved until orientation was achieved regardless of field strength, a principle we denote "orientation before destruction."

NMR refinement and peptide folding using the GROMACS software.

Nuclear magnetic resonance spectroscopy is used routinely for studying the three-dimensional structures and dynamics of proteins and nucleic acids. Structure determination is usually done by adding restraints based upon NMR data to a classical energy function and performing restrained molecular simulations. Here we report on the implementation of a script to extract NMR restraints from a NMR-STAR file and export it to the GROMACS software. With this package it is possible to model distance restraints, dihedral restraints and orientation restraints. The output from the script is validated by performing simulations with and without restraints, including the ab initio refinement of one peptide.

Reproducibility in the unfolding process of protein induced by an external electric field.

The dynamics of proteins are crucial for their function. However, commonly used techniques for studying protein structures are limited in monitoring time-resolved dynamics at high resolution. Combining electric fields with existing techniques to study gas-phase proteins, such as single particle imaging using free-electron lasers and gas-phase small angle X-ray scattering, has the potential to open up a new era in time-resolved studies of gas-phase protein dynamics. Using molecular dynamics simulations, we identify well-defined unfolding pathways of a protein, induced by experimentally achievable external electric fields. Our simulations show that strong electric fields in conjunction with short-pulsed X-ray sources such as free-electron lasers can be a new path for imaging dynamics of gas-phase proteins at high spatial and temporal resolution.