Protein structure prediction models are now capable of generating accurate 3D structural hypotheses from sequence alone. However, they routinely fail to capture the conformational diversity of dynamic biomolecular complexes, often requiring heuristic MSA subsampling approaches for generating alternative states. In parallel, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool for imaging near-native structural heterogeneity, but is challenged by arduous pipelines to transform raw experimental data into atomic models. Here, we bridge the gap between these modalities, combining cryo-EM density maps with the rich sequence and biophysical priors learned by protein structure prediction models. Our method, CryoBoltz, guides the sampling trajectory of a pretrained biomolecular structure prediction model using both global and local structural constraints derived from density maps, driving predictions towards conformational states consistent with the experimental data. We demonstrate that this flexible yet powerful inference-time approach allows us to build atomic models into heterogeneous cryo-EM maps across a variety of dynamic biomolecular systems including transporters and antibodies.

Cryo-electron microscopy (cryo-EM) is a powerful technique for determining high-resolution 3D biomolecular structures from imaging data. Its unique ability to capture structural variability has spurred the development of heterogeneous reconstruction algorithms that can infer distributions of 3D structures from noisy, unlabeled imaging data. Despite the growing number of advanced methods, progress in the field is hindered by the lack of standardized benchmarks with ground truth information and reliable validation metrics. Here, we introduce CryoBench, a suite of datasets, metrics, and benchmarks for heterogeneous reconstruction in cryo-EM. CryoBench includes five datasets representing different sources of heterogeneity and degrees of difficulty. These include conformational heterogeneity generated from designed motions of antibody complexes or sampled from a molecular dynamics simulation, as well as {compositional heterogeneity from mixtures of ribosome assembly states or 100 common complexes present in cells. We then analyze state-of-the-art heterogeneous reconstruction tools, including neural and non-neural methods, assess their sensitivity to noise, and propose new metrics for quantitative evaluation. We hope that CryoBench will be a foundational resource for accelerating algorithmic development and evaluation in the cryo-EM and machine learning communities. Project page: https://cryobench.cs.princeton.edu.

Cryo-electron microscopy (cryo-EM) is an experimental technique for protein structure determination that images an ensemble of macromolecules in near-physiological contexts. While recent advances enable the reconstruction of dynamic conformations of a single biomolecular complex, current methods do not adequately model samples with mixed conformational and compositional heterogeneity. In particular, datasets containing mixtures of multiple proteins require the joint inference of structure, pose, compositional class, and conformational states for 3D reconstruction. Here, we present Hydra, an approach that models both conformational and compositional heterogeneity fully ab initio by parameterizing structures as arising from one of K neural fields. We employ a hybrid optimization strategy and demonstrate the effectiveness of our approach on synthetic datasets composed of mixtures of proteins with large degrees of conformational variability. We additionally demonstrate Hydra on an experimental dataset imaged of a cellular lysate containing a mixture of different protein complexes. Hydra expands the expressivity of heterogeneous reconstruction methods and thus broadens the scope of cryo-EM to increasingly complex samples.