https://www.biorxiv.org/content/10.1101/2023.08.17.553673v1

In this manuscript, we show that the structure of a gas phase protein can be determined at near- atomic (2.6 Å) resolution by cryo-EM. To make this possible we developed a workflow with bespoke hardware: native electrospray ion-beam deposition (ESIBD), consisting of native electrospray ionization, mass selection, gentle landing of the protein ions, in-situ ice embedding, and clean cryo- transfer.

we demonstrate the following key points:

• We present a 2.6 Å cryo-EM density map of the tetrameric protein complex β-galactosidase. This resolution allows us to determine and analyze structural changes on the side-chain level.

• We identify a single dominating gas-phase structure which retains the native secondary and tertiary structure to a high degree.

• We observe compaction of the tetrameric complex, due to closing of cavities and grooves upon removal of water.

• MD simulations identify dehydration as the main driver of the observed structural changes.

This year we are very active in showing recent results: online and in person.

- Protein Society Webinar: Youtube Video

- ASMS meeting

- GRC Dynamics at Surfaces

- IMC20 (international microscopy conference)

- MS+M_23

We have modified a commercial mass spectrometer for the native deposition of large, native protein complexes. To this end, we added a landing module, and modified the vacuum system to increase transmission. The new instrument has a narrow beam energy distribution for gentle deposition. It can produce mass-selected samples for AFM and TEM from native protein ion beams. With the instrument, we demonstrate the non-covalent protein complex ADH retains enzymatic activity after deposition and rehydration.

For the full article, click here.

Our recent preprint, Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition, has now been published in PNAS Nexus. Click here for the full article.

×

In our latest preprint, Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition, we introduce native electrospray ion-beam deposition (native ES-IBD), a mass-selective sample preparation workflow for cryo-EM. Folded protein ions are generated by native electrospray ionization, mass-filtered, and gently deposited on cryo-EM grids, which are subsequently frozen in liquid nitrogen. Native ES-IBD has the potential to complement and accelerate established cryo-EM sample preparation workflows, by direct correlation between cryo-EM maps with precise information on composition and conformation. We show 3D reconstructions of protein assemblies purified in the gas-phase.