Modeling Atomic Conformational Ensembles of Proteins via Test-Time Supervision of Boltz-2 on Cryo-EM Density Maps
Authors: Jay Shenoy, Miro Astore, Axel Levy, Frédéric Poitevin, Sonya M. Hanson, Gordon Wetzstein
Abstract
Knowledge of a protein's atomic conformational ensemble is critical to determining its function, yet state-of-the-art ensemble prediction models are limited by lack of high-quality conformational data from simulation or experiment. Recent advances in heterogeneous reconstruction for cryo-electron microscopy (cryo-EM) have enabled scientists to visualize ensembles of density maps for larger proteins and complexes not typically accessible through simulation, but building atomic models into these maps remains a challenge. Traditionally, ensemble prediction models are trained via a two-stage process: experimental density maps are converted into atomic structural ensembles through model building, after which these structures are used to train sequence-to-atomic ensemble predictors. In this work, we propose a new principle for fine-tuning pre-trained static structure prediction models such as Boltz-2 directly on raw cryo-EM maps, bypassing the two-stage process. We apply this technique to the problem of atomic model building by fine-tuning Boltz-2 to generate atomic conformations from an input ensemble of cryo-EM maps, achieving superior model building accuracy compared to prior work. Beyond overfitting to individual map ensembles, our method, CryoSampler, also shows preliminary evidence of in-domain generalization after fine-tuning, sampling diverse atomic conformations for an unseen sequences within the same protein family without requiring cryo-EM data. These capabilities indicate that CryoSampler holds the potential to train next-generation atomic ensemble prediction models directly on raw cryo-EM measurements.
Protein automodeling from cryo-EM density maps faces unique challenges in enforcing physicochemical validity and managing conformational heterogeneity. Current solvers are often limited to static predictions or require computationally intensive heuristic searches. We present CryoACE, an end-to-end framework that reconstructs precise atomic graphs for both homogeneous and heterogeneous structures. Our method features two key innovations: an atom-centric reconstruction paradigm, where density features are sampled directly at atomic coordinates and iteratively recycled to refine structures, replacing expensive voxel convolutions for efficient multimodal fusion; and a training-free guidance mechanism that leverages predicted local resolution priors to resolve dynamic ambiguity. Validated on a newly constructed high-quality dataset, CryoACE significantly outperforms existing baselines on static benchmarks and, for the first time, unveils atomic-level dynamic conformations on complex real-world datasets like EMPIAR-10345 without relying on pre-built static structures.
Despite the growing availability of cryo-electron microscopy (cryo-EM) density maps, effectively leveraging them for protein representation remains challenging. First, current methods lack a general-purpose protein pretraining framework tailored for cryo-EM density maps, designed for protein-related property prediction. Second, existing approaches typically partition density maps into local box regions and model them independently, overlooking interactions across boxes which are essential for capturing global structural context in cryo-EM density map. To address these challenges, we propose CryoProt, a protein pretraining framework designed for cryo-EM density maps. CryoProt introduces a Map Encoder based on multi-head latent attention (MLA), where box-level representations interact through a shared latent space, enabling explicit modeling of cross-box dependencies within the density map. Furthermore, we adopt a multi-task pretraining strategy to learn generalizable representations that can be effectively transferred to diverse downstream tasks, such as protein flexibility prediction, where cryo-EM density maps are not required and can be inferred implicitly by the pretrained model. Experimental results demonstrate that CryoProt consistently outperforms existing state-of-the-art methods across multiple benchmarks, achieving up to 12% improvement over the best-performing baselines, highlighting the importance of modeling cross-box interactions in cryo-EM data. The source code is publicly available at https://anonymous.4open.science/r/CryoProt.
Generative models trained on public databases of protein structures, most of which have been determined by X-ray crystallography, now provide powerful priors for structure prediction. However, they are not readily conditioned on the measurements from a new crystallographic experiment, limiting their use for X-ray structure determination. In crystallography, the measured structure-factor amplitudes do not by themselves determine an electron density map or atomic structure because the associated phases are unobserved and must be inferred. Structure determination therefore remains an inverse problem in which candidate models must be both structurally plausible and consistent with measured diffraction data, often requiring substantial manual refinement by human experts. Emerging methods aim to incorporate experimental information more directly into predictive and refinement workflows. We present CrystalBoltz, a generative framework that casts crystallographic refinement as Bayesian inference over atomic structures and operates directly on structure-factor amplitudes. CrystalBoltz moves from unguided generation with a pre-trained prior over protein structures to experiment-guided posterior sampling, followed by atomic coordinate and B-factor refinement. Across multiple protein crystallography datasets, CrystalBoltz attains lower coordinate RMSD and lower R-factors than the strongest baselines considered, while reducing runtime by a factor of 33 relative to existing experimentally guided refinement.