Stephan Rauschenbach | Instruct Biennial Structural Biology Conference 2024

[@wolpumba4099]

*Combining Mass Spectrometry and Electron Microscopy for Enhanced Molecular Imaging* * *0:00:18** Introduction:* The speaker introduces the concept of merging mass spectrometry, specifically electrospray ionization mass spectrometry, with high-resolution microscopy techniques like electron microscopy. * *0:00:38** Historical Context:* The speaker draws a parallel between the work of Robert Hooke in microscopy and the challenges faced in modern sample preparation. * *0:02:53** Challenges in Scanning Probe Microscopy (SPM):* SPM, despite achieving atomic resolution, requires ultra-high vacuum conditions and thermal sublimation, limiting its application to biological samples. * *0:04:44** Electrospray Ionization as a Solution:* Electrospray ionization is presented as a method to generate intact gas-phase ions of biological molecules, overcoming the limitations of SPM. * *0:05:34** Controlled Ion Deposition:* The speaker explains the precise control over ion deposition achieved through mass filtering, beam focusing, and energy adjustment, allowing for controlled sample preparation. * *0:07:15** Application to Glycans:* The technique is demonstrated on glycans, showing its ability to image branching and connectivity, overcoming the limitations of traditional mass spectrometry in analyzing these complex molecules. * *0:10:55** Atomic Resolution with AFM:* Non-contact Atomic Force Microscopy (AFM) with a carbon monoxide tip is shown to achieve atomic resolution on glycans, revealing details of sugar subunits. * *0:13:04** Atomic Resolution in Cryo-EM:* The speaker highlights the capability of cryo-electron microscopy (cryo-EM) to achieve atomic resolution in both material science and biological applications. * *0:14:12** Challenges in Cryo-EM Sample Preparation:* The speaker discusses the complexities of cryo-EM sample preparation, particularly the blotting and plunge-freezing process, and proposes native electrospray ion beam deposition as an alternative. * *0:16:19** Investigating Gas-Phase vs. Solution Structures:* The central question is whether structures observed in the gas phase (mass spectrometry) are representative of those in solution (cryo-EM or crystallography). * *0:17:35** Instrument Design:* A custom-built instrument is described, combining a modified orbitrap with an ion beam column and a specialized landing system for controlled deposition onto cold TEM grids. * *0:18:49** Initial Results:* Preliminary data from room-temperature deposition onto amorphous carbon grids show promising results, with the rough shape of beta-galactosidase retained. * *0:19:56** Optimizing Ion Beam Conditions:* The speaker emphasizes the importance of optimizing ion beam conditions to minimize energy spread and ensure gentle treatment of the particles. * *0:22:26** Sample Transfer and Substrate Considerations:* A specialized transfer system is described to maintain sample integrity during transfer to the cryo-EM, and the choice of amorphous carbon as a substrate is discussed. * *0:24:45** Molecular Dynamics Simulations:* Simulations are used to model the collision of proteins with a graphene surface, demonstrating that the gas-phase fold is retained at low collision energies. * *0:26:05** Ice Embedding:* The ability to grow ice in a controlled manner on the sample is demonstrated, offering a potential matrix for cryo-EM studies. * *0:26:47** High-Resolution Structures from Deposited Beta-Galactosidase:* 2.5 Angstrom resolution structures of deposited beta-galactosidase are obtained, revealing a slightly more compact structure compared to the solution structure due to dehydration. * *0:29:22** Molecular Dynamics Simulations of Dehydration:* Simulations show that the removal of water molecules leads to a compaction of the protein structure, consistent with the experimental observations. * *0:31:03** Outlook and Future Directions:* The speaker presents preliminary results on other proteins, including GroEL, C-reactive protein, and GDH, and discusses ongoing work with membrane proteins. * *0:32:46** Conclusion:* Electrospray ion beam deposition is presented as a viable technique for cryo-EM sample preparation, offering unique insights into protein structure and the effects of dehydration. I used gemini-1.5-pro-exp-0827 on rocketrecap dot com to summarize the transcript. Cost (if I didn't use the free tier): $0.06 Input tokens: 40975 Output tokens: 916