Periodic Reporting for period 1 - PNMS (Extending the applicability of Cryo-EM for fragile biological systems via ultra-pure cryo-samples from Preparative Native Mass Spectrometry)
Reporting period: 2020-06-01 to 2022-05-31
Understanding and controlling the function of biological macromolecules, requires detailed information on their structure, including conformation, ligands, flexibility, and stability. This information can help to reveal causes and cures for diseases. For example, G-protein coupled receptors (GPCRs) are a large and heterogeneous group of membrane proteins that mediate cellular response to hormones and neurotransmitters. They are a major target for the development of treatments of cardiovascular and gastrointestinal diseases.
Cryo electron microscopy (cryo-EM) has become the method of choice to obtain high (often atomic) resolution structures of protein complexes that are not amenable to alternative techniques like X-ray crystallography or nuclear magnetic resonance. Despite major advances, sample preparation is typically the main bottleneck of the cryo-EM workflow. Challenges include denaturation of proteins at the air-water interface, sample heterogeneity, and inhomogeneous ice-thickness. All of these effects can decrease resolution and thus hide structural information.
Complementary information, in particular from mass spectrometry (MS) based techniques can help to find optimal sample conditions, interpret and refine 3D structures, reveal native binding sites and strength, and provide information on small ligands and flexible protein regions. By combining native MS and electrospray ion-beam deposition (ES-IBD) into a novel workflow, termed native ES-IBD, the current project aimed at making preparation of cryo-EM samples of protein complexes more reliable and selective and allow for unambiguous assignment of complementary information from mass spectrometry to high-resolution structures from cryo-EM.
Using native ES-IBD and cryo-EM, mass-selected and ice-free samples were prepared and imaged, demonstrating high contrast as well as control over particle distribution, deformation, and dissociation. Corresponding 2D classes and 3D EM density maps show that the overall shape of protein assemblies is largely preserved. Small structural changes due to dehydration, landing, or surface interaction limit resolution. Instrumentation was developed to control temperature and hydration which may allow to overcome these limitations in the future. The results imply that the native ES-IBD may lead to an acceleration of drug development if the current limitations can be overcome.
Cryo electron microscopy (cryo-EM) has become the method of choice to obtain high (often atomic) resolution structures of protein complexes that are not amenable to alternative techniques like X-ray crystallography or nuclear magnetic resonance. Despite major advances, sample preparation is typically the main bottleneck of the cryo-EM workflow. Challenges include denaturation of proteins at the air-water interface, sample heterogeneity, and inhomogeneous ice-thickness. All of these effects can decrease resolution and thus hide structural information.
Complementary information, in particular from mass spectrometry (MS) based techniques can help to find optimal sample conditions, interpret and refine 3D structures, reveal native binding sites and strength, and provide information on small ligands and flexible protein regions. By combining native MS and electrospray ion-beam deposition (ES-IBD) into a novel workflow, termed native ES-IBD, the current project aimed at making preparation of cryo-EM samples of protein complexes more reliable and selective and allow for unambiguous assignment of complementary information from mass spectrometry to high-resolution structures from cryo-EM.
Using native ES-IBD and cryo-EM, mass-selected and ice-free samples were prepared and imaged, demonstrating high contrast as well as control over particle distribution, deformation, and dissociation. Corresponding 2D classes and 3D EM density maps show that the overall shape of protein assemblies is largely preserved. Small structural changes due to dehydration, landing, or surface interaction limit resolution. Instrumentation was developed to control temperature and hydration which may allow to overcome these limitations in the future. The results imply that the native ES-IBD may lead to an acceleration of drug development if the current limitations can be overcome.
A custom deposition stage, controlled using a home-built software, was added to a commercial mass spectrometer (Thermo Scientific Q Exactive UHMR). The modified instrument allows to produce intense ion-beams of native proteins and protein complexes for deposition onto TEM grids and graphite samples. Beam intensity, total beam energy, and beam energy distribution were characterized and optimized. Mass-selective, clean, protein samples with consistent quality can typically be prepared in less than 30 minutes. Soft-landed samples of protein complexes in a mass range from 150 to 800 kDa were imaged using atomic force microscopy (AFM), negative stain TEM, and cryo-EM. For cryo-EM, 2 nm amorphous carbon films were identified as ideal substrate due to high contrast and suppression of thermal diffusion. The results show that protein shapes remain in line with known native protein structures when limiting landing energies to below 10 eV per charge. 2D classes and 3D EM density maps from soft-landed samples show that proteins remain folded and subunits in protein complexes remain attached, confirming retention of near-native structures of large protein complexes in the gas-phase. The resolution did not allow for determination of secondary structure. A series of control experiments suggest that preparation of protein solutions for mass spectrometry, microscope settings, preferred orientation, beam-induced motion, are not limiting. Instead, our results indicate that heterogeneity in the secondary and tertiary structure, introduced by dehydration, radiation damage, landing, or surface interactions, limits the amount of information that can be obtained by averaging techniques.
A cryogenically cooled landing stage was implemented to control temperature and hydration throughout the workflow. It forms the basis for variations of the workflow that include controlled rehydration, which is essential to retain or regain native solution phase structures. Unfortunately, COVID-19 related delays in the delivery of essential parts delayed completion until shortly after the end of the project.
The results were presented at the conference for Isolated Biomolecules and Biomolecular Interactions (IBBI) in 2022. Further disseminations shortly after the end of the project include talks at the International Mass Spectrometry Conference (IMSC) 2022, the Faraday discussion on “Challenges in biological cryo electron microscopy” in 2022, and the workshop “Frontiers in Native Mass Spectrometry and Single-Molecule Imaging”, co-organized by the researcher. A preprint focusing on general instrument performance and applications as posted to arxiv and submitted to a high-impact journal. It is, as of July 2022, in minor revision. A publication on the specific use for cryo-EM sample preparation has been accepted by PNAS Nexus. A third paper focusing on the choice of the substrate and surface induced dissociation has been accepted as part of the Faraday discussion mentioned above. Thermo Fisher Scientific and the University of Oxford have filed a joint patent application concerning variations of the workflow beyond the action. The close collaboration with Thermo Fisher Scientific on this project resulted in a unique opportunity for the fellow to join the company while being able to complete construction and characterization of the cryo landing stage at the University of Oxford.
A cryogenically cooled landing stage was implemented to control temperature and hydration throughout the workflow. It forms the basis for variations of the workflow that include controlled rehydration, which is essential to retain or regain native solution phase structures. Unfortunately, COVID-19 related delays in the delivery of essential parts delayed completion until shortly after the end of the project.
The results were presented at the conference for Isolated Biomolecules and Biomolecular Interactions (IBBI) in 2022. Further disseminations shortly after the end of the project include talks at the International Mass Spectrometry Conference (IMSC) 2022, the Faraday discussion on “Challenges in biological cryo electron microscopy” in 2022, and the workshop “Frontiers in Native Mass Spectrometry and Single-Molecule Imaging”, co-organized by the researcher. A preprint focusing on general instrument performance and applications as posted to arxiv and submitted to a high-impact journal. It is, as of July 2022, in minor revision. A publication on the specific use for cryo-EM sample preparation has been accepted by PNAS Nexus. A third paper focusing on the choice of the substrate and surface induced dissociation has been accepted as part of the Faraday discussion mentioned above. Thermo Fisher Scientific and the University of Oxford have filed a joint patent application concerning variations of the workflow beyond the action. The close collaboration with Thermo Fisher Scientific on this project resulted in a unique opportunity for the fellow to join the company while being able to complete construction and characterization of the cryo landing stage at the University of Oxford.
Native ES-IBD represents a significant improvement compared to the previous state of the art, which was limited by the used of stain, low ion-beam intensity, and low control over landing energy. Direct images with clear shapes confirm that proteins can remain folded and subunits can remain attached despite dehydration, collision with the substrate, and prolonged exposure to the substrate-vacuum interface at room temperature. The mass-spectrometric information, selectivity, and strong contrast can be useful for screening and interpreting higher resolution structures obtained using conventional cryo-EM. Further, native ES-IBD can provide complementary information to help address the challenges of the conventional plunge-freezing workflow. There may be great potential in eliminating solvent and ice-related effects, including denaturation at the air-solvent interface, strong and inhomogeneous background signal, unintentional devitrification, beam-induced motion of the ice, and inhomogeneous particle distribution.
The unique instrumentation developed as part of this project has the potential to greatly improve cryo-EM sample preparation. The high level of reproducibility and control implies a large potential to reduce time and cost while maintaining near native structure. The project showed that these objectives are closer to being realized than previously anticipated by the adjacent communities.
As the approach is based on a widely available commercial instrument, it can be adopted by other labs with reasonable effort. Thus, applications can be scaled up quickly and have the potential to significantly accelerate drug development.
The unique instrumentation developed as part of this project has the potential to greatly improve cryo-EM sample preparation. The high level of reproducibility and control implies a large potential to reduce time and cost while maintaining near native structure. The project showed that these objectives are closer to being realized than previously anticipated by the adjacent communities.
As the approach is based on a widely available commercial instrument, it can be adopted by other labs with reasonable effort. Thus, applications can be scaled up quickly and have the potential to significantly accelerate drug development.