Cryo-EM Structure Determination
SBD boasts an extensive track record in the production of large protein molecules and intricate complexes. Our expertise shines through in the successful preparation of CRISPR Cas12a in complex with target DNA and guide RNA. We've also prepared complexes of Cyclin D/Cdk4, COVID-19 N-protein/antibody, and PROTACS, effectively navigating the challenges of their intricate structures.
Our proficiency is exemplified by our achievement of high-resolution crystal structures for these protein complexes. However, recognizing the evolving landscape, we've swiftly embraced cryo-electron microscopy (cryo-EM) as a rapid and efficient avenue to attain 3D structures for sizable molecular targets with molecular weights exceeding 100 kDa.
Through a seamless transition, we've harnessed the power of cryo-EM to unravel the structural complexities of large protein molecules and their corresponding complexes. Notably, SBD remains a pioneering entity, uniquely equipped to offer a comprehensive suite of services encompassing protein production, activity study, X-ray crystal structure determination, and cryo-EM structure determination – all under one roof.
Our consolidated approach not only ensures a holistic understanding of your target but also underscores our commitment to innovation and precision within the field of structural biology.
Our general procedure to solve a cryo-EM structure is below:
1. Protein sample preparation: Expression and purification of the target protein via affinity, ion exchange and size-exclusion chromatographic methods.
2. Protein sample evaluation: Protein sample homogeneity will be evaluated by gel filtration chromatography profile, dynamic light scattering, and negative staining TEM to see the particle’s monodispersity, and to determine if the sample is qualified for single particle cryo-EM.
3. Sample Preparation for Cryo-EM: Prepare a thin layer of amorphous ice containing the protein sample on a specialized EM grid. Add a small amount of protein sample to the grid, blot off excess solution, and rapidly freeze and store the grid in cryogen.
4. Data Collection: Load the frozen cryo-EM grid into the electron microscope. The Titan Krios G3 and G4 are a widely used and respected cryo-EM system known for their high-resolution data collection capabilities. Collect a large number of images of individual particles using low-dose conditions to minimize radiation damage.
5. Structural determination: Image processing using MotionCor2 and cryoSPARC, and 3D reconstruction will generate an initial 3D model at our lab. Model building and refinement will be fit and refined with Coot and real-space refinement to improve the fit between the model and the density map.
6. Structural report: SBD's team of scientists meticulously analyze the structures of your target, subsequently delivering a comprehensive interactive session encompassing 3D molecular graphics. This engaging presentation, spanning approximately 20 to 30 slides, delves into the structural insights, pivotal interactions, and noteworthy inhibitor-induced conformational alterations.
Furthermore, this presentation is accompanied by a live Q&A segment during either an in-person meeting or a virtual conference facilitated by screen sharing. Your queries and design considerations are addressed promptly and in real-time, ensuring that you receive the most relevant and up-to-date information.
It's worth noting that our team has achieved a remarkable level of client satisfaction, approaching nearly 100%. This is a testament to our dedication and proficiency in delivering valuable structural insights that meet and exceed our clients' expectations.