PNMS · Extending the applicability of Cryo-EM for fragile biological systems via ultra-pure cryo-samples from Preparative Native Mass Spectrometry
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2020-06-01 → 2022-05-31
- EU contribution
- €212,934
- Participants
- 1
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Extending the applicability of Cryo-EM for fragile biological systems via ultra-pure cryo-samples from Preparative Native Mass Spectrometry
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.
Data: CORDIS, © European Union
Project objective
Precise structural, conformational, as well as chemical information of biological molecules forms the basis for comprehension and control of biological processes, including metabolism, pathological processes, and drug targeting. Cryo-electron microscopy (cryo-EM) has evolved into one of the leading methods for structural characterisation of folded proteins and protein complexes reaching atomic resolution through averaging. Thus, the unambiguous assignment of thousands of images to different oligomers, conformers, and fragments is a key requirement for high-resolution single particle cryo-EM. However, reliable preparation of homogeneous cryo samples remains one of, if not the, most important challenge in cryo-EM. Native mass spectrometry (native MS) allows to retain proteins in a near native state in an ultrapure gas-phase molecular ion-beam, providing complementary information on mass, composition, conformation and ligand binding sites. Preparative mass spectrometry (prep-MS) is able to generate ultrapure samples by soft landing of mass- and conformation selected molecules on surfaces for subsequent analysis. Here, we propose to implement preparative native mass spectrometry (pnMS), combining the strengths of native MS, prep-MS, and cryo-EM, to establish a new versatile pathway for comprehensive structural analysis of biological systems. We will design ion optics to couple a mass selected molecular ion beam from a commercial high-performance MS and a landing stage to add deposition capability. Initial experiments will focus on benchmark systems such as BSA and GroEL, before approaching more challenging proteins, including heat shock proteins (HSPs) and G-Protein coupled receptors (GPCRs). Ultimately, pnMS should enable the structural biology community to obtain a comprehensive understanding of structural variety (proteoforms), protein interactions (interactome), and synthetic structures, e.g. needed for customised medicine.
Original text from CORDIS.
Participants
- THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD · OxfordCoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union