# The Resolution Revolution Is Still Ongoing **Author:** CryoSPARC Team **Published:** May 27, 2026 **Category:** Small Protein Targets **Tags:** structural-biology, cryosparc, cryo-em, small-protein **Summary:** High-Resolution Ab-Initio Reconstruction: Extending Cryo-EM to Smaller Particles with CryoSPARC™ **Source:** https://cryosparc.com/blog/high-resolution-ab-initio-reconstruction --- Over the past two decades, cryo-EM has revolutionized structural biology, enabling high-resolution structure determination of macromolecules and protein complexes that were previously inaccessible. By capturing multiple conformational states from a single dataset, cryo-EM has provided unique insights into molecular heterogeneity and dynamics. Despite this progress, important limitations remain. One long-standing challenge is the structural determination of small proteins, typically below ~50 kDa. In a recent [preprint](https://www.biorxiv.org/content/10.1101/2025.09.08.674935v1), Kookjoo Kim, Huan Li, and Oliver B. Clarke challenge this size limit by implementing a new data-processing workflow in [CryoSPARC](https://cryosparc.com/), termed high-resolution heterogeneous ab initio reconstruction (HR-HAIR). ![Figure1-HR-HAIR.png](https://cryosparc.com/images/blog/high-resolution-ab-initio-reconstruction/Figure1-HR-HAIR.png) ### **The Challenge of Small Proteins: Why Bother?** In many structural biology pipelines, cryo-EM is simply reserved for large protein complexes, while smaller proteins are redirected to complementary techniques such as X-ray crystallography. While pragmatic, this division overlooks key advantages that cryo-EM brings to the study of small enzymes and regulatory proteins. Cryo-EM enables the direct observation of sample composition with minimal manipulation. How often are monomers, dimers, or transient subcomplexes discarded during data processing without a second look? And how often might these “minor” species be central to biologically or therapeutically relevant mechanisms of association, dissociation, or regulation? **Access to structures of small particles can therefore inform mechanism, target engagement, and assembly equilibria, critical parameters in drug discovery and development pipelines.** ![Figure2-HR-HAIR.png](https://cryosparc.com/images/blog/high-resolution-ab-initio-reconstruction/Figure2-HR-HAIR.png) ### **Acting on the Workflow: Focusing on Data Processing** When sample optimization for small particles and hardware improvements fail (or are too time-consuming to justify in a production environment) the natural next step is to focus on the third pillar of the cryo-EM workflow: data processing. This is the approach taken by Kookjoo Kim, Huan Li, and Oliver B. Clarke in their recent preprint. The authors identified a critical bottleneck in SPA of small proteins: the estimation of initial particle orientations during *ab initio* reconstruction. At low resolution, small proteins often lack sufficient structural features for reliable alignment, raising a simple but powerful question posed by Kim et al.: what happens if [ab initio reconstruction](https://guide.cryosparc.com/processing-data/all-job-types-in-cryosparc/3d-reconstruction/job-ab-initio-reconstruction) is run using high spatial frequency information? Their answer is **high-resolution heterogeneous ab initio reconstruction (HR-HAIR), a CryoSPARC-based workflow that derives initial particle orientations from high-frequency signal.** By incorporating higher resolution information from the start, increasing the number of iterations, and using very small resolution step sizes between iterations, HR-HAIR bypasses the need for reliable low-resolution features and converges on interpretable initial maps. **Using this approach, the authors demonstrated that high-resolution *ab initio* reconstruction can enable structural determination of proteins below 30 kDa.** The workflow was validated on several publicly available datasets, resolving iPKAc (39 kDa) to 2.7 Å and a hemoglobin αβ dimer (29 kDa) to ~4 Å. Notably, a 37 kDa Aca2–RNA complex was reconstructed directly from a blob-picked particle stack in a single HR-HAIR run, followed by local refinement, without prior 2D or 3D classification. These workflow highlights how software advances alone can extend the practical reach of cryo-EM. > *The fact that ab initio reconstruction alone is able to generate interpretable maps for iPKAc and Aca2-RNA, even in the presence of significant preferred orientation, suggests that the use of this approach for high resolution reconstruction and classification may be currently underestimated [...]* > ![Figure3-HR-HAIR.png](https://cryosparc.com/images/blog/high-resolution-ab-initio-reconstruction/Figure3-HR-HAIR.png) One important limitation, however, is that CryoSPARC’s Ab-Initio Reconstruction does not perform half-set splitting during this stage, leaving subsequent local refinement susceptible to overfitting and resolution overestimation. ### The New **Homogeneous Ab-Initio Refinement (BETA)** Building on these findings, [CryoSPARC v5](https://cryosparc.com/updates/v5.0) introduces a new reconstruction strategy directly inspired by [the HR-HAIR method](https://www.biorxiv.org/content/10.1101/2025.09.08.674935v1). **The new [Homogeneous Ab-Initio Refinement (BETA)](https://guide.cryosparc.com/processing-data/all-job-types-in-cryosparc/3d-reconstruction/job-homogeneous-ab-initio-refinement-beta?q=downsample), job adapts CryoSPARC’s [stochastic gradient descent](https://www.nature.com/articles/nmeth.4169)–based *ab initio* algorithm into a gold-standard refinement framework** that preserves the independence of the two half-sets and corresponding half-maps throughout reconstruction. ![Figure4-HR-HAIR.png](https://cryosparc.com/images/blog/high-resolution-ab-initio-reconstruction/Figure4-HR-HAIR.png) This job is streamlining the workflow for challenging datasets, where CryoSPARC’s stochastic gradient descent algorithm outperforms the traditional [expectation maximization](https://guide.cryosparc.com/expectation-maximization-in-cryo-em) algorithm used in refinements, and ensures an easier resolution estimation and [validation](https://guide.cryosparc.com/processing-data/all-job-types-in-cryosparc/post-processing/job-validation-fsc). By expanding cryo-EM into the small-particle regime, CryoSPARC v.5 enables gold standard workflows, where revisiting existing data can reveal structures, mechanisms, and opportunities that were previously missed.