Combining native protein mass spectrometry with serial electron diffraction to solve atomic structures of mass selected macromolecules

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We plan to add an electron diffraction component to a native protein mass spectrometer to create a new instrument that can derive atomic structures of macromolecules such as proteins. The key innovation is the use of superfluid helium droplets for sample cooling thereby effective field induced orientation and alignment. Mass and conformation selected proteins from a native electrospray ionization mass spectrometer are embedded in superfluid helium droplets, and in a pulsed electric field and elliptically polarized laser field, due to the permanent and induced dipoles of the protein, all three Euler angles of the protein can be precisely defined. The large polarizability volume of macromolecules (not the permanent dipole moment) and the low rotational temperature of the embedded macromolecules are the two elements of the “molecular goniometer”: changing the polarization properties of the laser field changes the orientation of the macromolecule. Electron diffraction patterns from macromolecule-doped droplets, one molecule per droplet, all oriented in the same direction, are accumulated with each successive pulse, until a satisfactory signal-to-noise ratio is achieved. Ultimately from the diffraction patterns of all orientations of the chosen macromolecule, the electrostatic potential is derived using the oversampling method for iterative phase retrieval and structure determination. In the past few years, we have accumulated preliminary data on electron diffraction of small molecules and cationic molecular clusters embedded in superfluid helium droplets, and on doping macromolecular ions into superfluid helium droplets. The next phase of the project is to construct a complete instrument to demonstrate the principle of the concept. We now can solve structures of nanocrystals embedded in superfluid helium droplets, both neutral and charged, without sample alignment. Therefore the background issue of the enclosing helium and the particle density issue of charged species are no longer major concerns. Our demonstrated resolution from pyrene dimer cations is 0.5 Å. Moreover, we have succeeded in doping macromolecular ions into superfluid helium droplets using a standard electrospray ionization source. Our accomplishments so far have laid the foundation for the next phase of progress, and we are now ready to demonstrate the principle of the concept. With the acquisition of a new electron gun, a upgrade to a native protein ion source, and a direct electron detector, we have a detailed plan to align all three pulsed beams, the laser beam, the ion doped droplet beam, and the electron beam, to obtain diffraction patterns of field aligned macromolecules. Our ultimate goal is to resolve atomic structures of mass and conformation selected macromolecules with 1 Å resolution from mixtures of protein solutions, microfluidic reactors, or labeled cells for proteins and protein complexes. The final instrument will reshape the landscape of structural biology, transform structure-based drug screening, and rapidly determine effects of mutations and deletions on structure. It will also offer structural assessment of components in polydisperse mixtures of nanomaterials important for biomedical applications. To mitigate the risks, we have recruited a specialist in mass spectrometry, Dr. David Russell, to be our consultant, and a specialist in data processing, Dr. Peter Schwander, to be a member of our team.