Acta Crystallographica Section D Structural Biology

Accurate determination of state occupancies is essential for interpreting the structural heterogeneity inherent in time-resolved crystallography. However, in cases of high spatial overlap between states, as commonly observed in time-resolved crystallography data, the strong correlation between occupancy and atomic displacement parameters (ADPs) can render single point estimates from standard refinement protocols unreliable. We introduce MEROS (Multi-state Ensemble Refinement for Occupancy Statistics), a pipeline that implements an ensemble refinement approach to assess the post-refinement occupancy-ADP statistics of multiple overlapping states. MEROS utilizes a Monte Carlo sampling of the parameter space, performing independent refinements from randomized starting occupancies and ADP values to empirically characterize the convergence and uncertainty of the solution. The method is implemented as a modular Python pipeline that wraps established refinement programs, ensuring compatibility with existing workflows. We demonstrate its applicability in two case studies: a two-state ligand binding model in T4 lysozyme L99A and a four-state covalent catalysis mechanism in {beta}-lactamase CTX-M-14. MEROS provides occupancy and ADP mean values with standard deviations that directly quantify the informational content of the experimental diffraction data.

Cryogenic electron microscopy (cryoEM) is now routinely used for high-resolution structure determination of biological macromolecules. However, many biological specimens exhibit varying degrees of preferred orientation on cryoEM grids, resulting in uneven sampling of three-dimensional Fourier space. This orientation bias produces anisotropic reconstruction artifacts and, in severe cases, can exacerbate particle misalignment during iterative refinement, thereby limiting the success rate of near-atomic resolution cryoEM structure determination. This protocol provides a practical guide for applying spIsoNet, a self-supervised deep-learning method, to mitigate preferred-orientation issues in cryoEM reconstructions. We describe two complementary workflows: (1) map Anisotropy Correction to correct anisotropic artifacts of cryoEM maps and (2) particle Misalignment Correction, which integrates spIsoNet with RELION external reconstruction to improve particle-pose estimation. We demonstrate these workflows using two influenza hemagglutinin (HA) trimer datasets representing moderate and severe degrees of preferred-orientation bias. The protocol includes installation instructions, parameter-selection guidance, quality-control checkpoints and troubleshooting advice, and can typically be completed in ~7 hours on a workstation equipped with four NVIDIA A100 GPUs. Together, these workflows provide step-by-step guidance for using the open-source spIsoNet software to mitigate the preferred-orientation problem directly from experimental data.

Structure elucidation of biological macromolecules by single particle cryogenic electron microscopy (SPA cryo-EM) or cryogenic electron tomography (cryo-ET) relies on low-dose imaging on cryogenic transmission electron microscopes (cryo-TEMs). Routine microscope setup remains technically demanding and can be time-consuming, particularly for inexperienced or infrequent users. We present LowDoseWizard, a guided workflow implemented in SerialEM that enables rapid and standardised setup of cryo-TEM imaging conditions. From minimal user input, the workflow configures microscope optics, camera parameters and image shift settings for all low-dose imaging states, and guides the user through key daily alignment procedures including beam shift offset calibration, objective lens astigmatism correction and coma-free alignment. The workflow is organised into modular routines that can be executed sequentially or independently, while microscope-specific acquisition parameters are defined in editable configuration files, allowing flexible adaptation to different instruments without modification of the core scripts. Across user sessions on three microscopes at EMBL Heidelberg, the complete setup required on average less than 15 minutes. To assess whether predefined imaging conditions generated by the workflow are compatible with high-resolution data collection, we acquired apoferritin data on a 200 kV Glacios and a 300 kV Titan Krios. These datasets yielded reconstructions at 1.62 [A] and 1.09 [A] resolution, respectively, demonstrating that rapid, guided setup can support near-atomic and atomic-resolution single particle cryo-EM. LowDoseWizard lowers the barrier to robust cryo-TEM setup, reduces the time spent on routine parameter selection and alignment, and helps users focus on sample-specific aspects of data acquisition such as target selection. The workflow should be particularly valuable in shared instrumentation environments, where accessibility, reproducibility and efficient microscope use are critical.

Accurate determination of the image pixel size is critical for quantitative cryo-electron microscopy analyses, yet existing calibration methods remain under-utilized because installation barriers and workflow complexity discourage routine adoption. To fill in this gap, a web-based application, WebCalEM, was developed to transform specialized calibration procedures into an accessible routine practice. Micrographs of any specimen with a known crystalline lattice, such as gold or graphene-oxide, are uploaded through a standard browser, processed entirely client-side, and analyzed with real-time visualization and downloadable statistical outputs. The application is delivered as a single self-contained HTML file that runs in any modern web browser without server-side computation, a configuration well suited to isolated core-facility microscope workstations. Cross-standard consistency between gold and graphene-oxide measurements across two microscopes and ten magnification settings yields a Bland-Altman bias of -0.005% of nominal with 95% limits of agreement of [-0.30%, +0.29%]. By delivering this workflow with no local installation, WebCalEM lowers the practical barrier to documented per-dataset magnification calibration in routine cryo-EM operation. SynopsisWebCalEM is a browser-based, install-free application that performs routine cryo-EM pixel-size calibration directly from gold or graphene-oxide reflections in standard sample-support grids using sub-pixel Fourier-space peak localization; it reproduces the precision of established command-line calibration tools while removing the installation barrier and supporting retrospective per-region calibration on archived datasets.

Serial femtosecond crystallography (SFX) is increasingly employed to determine protein structures and study conformational dynamics under physiological conditions, but the effects of sample preparation and delivery on crystallized macromolecules remain poorly understood. Here, we report the analysis of soybean lipoxygenase-1 (SLO) microcrystals collected by SFX at the Linac Coherent Light Source. During data analysis, we observed unexpected polymorphism in SLOs unit-cell parameters, arising from two compounding factors: indexing ambiguities caused by the pseudo-tetragonal symmetry of the SLO crystal lattice, and true non-isomorphism between individual crystals driven by differential hydration of microcrystals embedded in a hydroxyethylcellulose carrier medium. By combining unit-cell clustering with systematic reindexing, we resolved two distinct polymorphs and determined independent structures from a single experiment. Although the two structures show little difference in the average atomic coordinates (average all-atom RMSD of 0.34 [A]), an approximately 8.5% difference in crystal solvent content produces measurable differences in crystal contacts and conformational flexibility. The more hydrated (large-cell) polymorph exhibits greater inter-domain flexibility and harmonic disorder in hydrophobic core residues belonging to the catalytic network of the enzyme. In the dehydrated (small-cell) polymorph, these same residues adopt discrete alternative conformations resolvable in the electron density. Our results demonstrate that sample delivery conditions in serial crystallography can significantly modulate the apparent conformational landscape of crystallized proteins, with direct implications for the interpretation of protein dynamics from SFX data. SynopsisUsing soybean lipoxygenase-1 (SLO) as a model system, we show that the carrier media used for sample delivery in SFX experiments can alter the solvent content of protein microcrystals, producing distinct crystal polymorphs within a single experimental sample. Although the average atomic coordinates are minimally perturbed, a detailed analysis of crystallographic displacement parameters reveals that conformational flexibility is sensitive to sample delivery conditions, a consideration of broad relevance for time-resolved SFX experiments that aim to capture functionally important protein motions.

Cryo-electron tomography is an expanding technology for the study of macromolecules, viruses, and cells. It is often applied to specimens that are too large or heterogeneous for methods based on 2D image averaging such as single particle analysis, e.g., intracellular membranes or organelles. Current practice records a tilt series of projection images in rotation. Reconstruction is normally an ill-posed mathematical problem. Particularly for the under-determined case of sparse data, discrete tilt angles, and a limited tilt range, characteristic artifacts appear in the reconstructed slices. Much of what appears as noise is in fact structural: the projection of contrast from different planes. Various schemes are employed to regularize the reconstruction, including machine-learning frameworks built on neural networks. To the extent that the noise is structural, it might be suppressed by deconvolution with a suitable kernel. This was demonstrated and has been used regularly in cryo-STEM tomography of thick specimens where the under-sampling problem is particularly acute. Here we present 3dcon as an open-source extension of the entropy-regularized deconvolution algorithm that had been adopted from fluorescence microscopy. It takes advantage of modern computing hardware for convenient and fast processing. Deconvolution is entirely algorithmic, meaning that successful processing of the data does not depend on the data itself. As such it should be robust in a wide variety of applications.

Although an increasing number of protein structures are determined by cryogenic electron microscopy (cryo-EM), protein structure modeling frequently suffers from residue misassignments and sequence register shifts, particularly in regions with ambiguous density. Here, we present DAQplugin, a ChimeraX plugin that performs real-time evaluation of protein models against cryo-EM density maps using the deep-learning-based residue-wise model quality (DAQ) score. Unlike existing validation tools that are typically applied after model construction, DAQplugin enables real-time deep-learning-based validation during model building and refinement. To our knowledge, DAQplugin is the first tool that provides real-time deep-learning based validation of protein models for cryo-EM map within an interactive modeling environment. In addition to identifying potential modeling errors, DAQplugin also provides guidance for correcting sequence register shifts by suggesting alternative residue placements along the backbone. The computation in this plugin is designed to run efficiently on general CPUs without requiring GPU hardware. Using DAQplugin, users can perform deep-learning-based validation on standard laptops during interactive model building, model-map fitting, and refinement. DAQplugin is able to facilitate more accurate interpretation of cryo-EM density maps and improve the reliability assessment of protein structure models. SynopsisDAQplugin provides real-time residue-wise validation of protein models with cryo-EM maps in ChimeraX.

Macromolecular crystallographic refinement underpins structural biology, yet existing software packages often lack accessible, modular codebases amenable to rapid method development. Here, we introduce TorchRef, a PyTorch-based crystallographic refinement framework that exposes all refinable parameters, atomic coordinates, displacement parameters, occupancies, and scale factors to automatic differentiation. The framework implements FFT-based structure-factor calculations, the French-Wilson treatment of intensities, bulk-solvent modeling with established mask parameters, and stereochemical restraints from the CCP4 Monomer Library. A modular target architecture allows loss functions to be combined, weighted, and extended independently of the core refinement machinery. Validation against 1,000 PDB structures demonstrates that TorchRef-based refinement reproduces a median R-free within 1% of Phenix while maintaining comparable model quality. Structure factor calculation in TorchRef scales readily across multiple CPU cores and is over 100 times faster on modern GPUs than CCTBX. To showcase how modern methods like time-resolved crystallography can benefit from the flexibility that TorchRef provides, we implemented direct refinement of a typical time-resolved model against amplitude differences, a use case currently not explored by classic refinement programs. TorchRef is released under the MIT license with full API documentation and tutorials, providing an accessible platform for developing and testing new crystallographic refinement protocols. SynopsisTorchRef is an open-source PyTorch-based crystallographic refinement framework that exposes all refinable parameters to automatic differentiation, delivers GPU-accelerated structure-factor evaluation more than 100x faster than CCTBX, and enables new workflows, such as direct refinement against amplitude differences in time-resolved crystallography.

Cryogenic-electron microscopy (cryo-EM) has revolutionized the field of protein structural biology. The structures of large membrane proteins are now routinely determined by cryo-EM to near atomic resolution. However, in the medium resolution range of cryo-EM maps (>[~]2 [A]), negatively charged sidechains of acidic residues are not well-resolved due to the negative electrostatic potential of the region. This may lead to incorrect sidechain models for residues like glutamic acid or aspartic acid that are central for proton transfer activity in various respiratory and photosynthetic enzymes. We previously proposed that the acidic residues with weak or non-existent cryo-EM density can be modeled to represent their low proton affinity conformations. Here, we tested this hypothesis on a larger data set of acidic amino acid residues in two high-resolution respiratory complex I structures. By using faster sidechain modeling and proton affinity prediction tools, we created a workflow that generates sidechain conformations of selected amino acid residues. We validated the sidechain conformation predictions by Q-score analysis and atomistic molecular dynamics simulations in different charged states. The proposed workflow provides a way to rapidly obtain sidechain conformations of acidic residues with weak cryo-EM densities and can be integrated into the existing cryo-EM modeling pipelines to speed up sidechain rotamer prediction.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN2 to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN2 refill system that is compatible with gas cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling.

In single particle cryo-EM, analysis of continuous conformational heterogeneity has always been challenging. Both linear and deep learning-based methods treat conformational heterogeneity as perturbations to the consensus average conformation, limiting their capability in analyzing large protein motions. While classic conformational classifications are capable of handling large domain motion, they bin continuous protein dynamics into discrete static substates. Here, we present cryoROLE, a computational tool that extracts the continuous conformational dynamics embedded in the static composite map constructed from multi-body refinement into a landscape of relative orientation between the moving domains. Depicted in real space, the landscape allows intuitive interpretations of domain motion and the population of poses in the conformational space. Applying it to various biological systems reveals hidden conformational dynamics that are relevant to protein functions.

Macromolecular crystallography is limited by the phase problem: diffraction experiments measure amplitudes but not the phases required to reconstruct electron density. Existing phasing routes usually seek enough continuous phase information for density modification and model building to converge. Here, we ask how much phase information can be discarded while preserving convergence. We analyzed 14,148 diffraction datasets from chiral crystals to characterize centric reflections in reciprocal-space asymmetric units. After conditioning by centric trace and, where required, index parity, the two theoretical symmetry-allowed phase values were populated near equally, close to 50:50, independent of space group, defining a compact symmetry scaffold. We then retained this exact scaffold while compressing reference acentric phases to a one-bit alphabet {0, {pi}}; as expected from their diffuse parent distribution, the assignments were also near-balanced. Although this binary representation, with fixed attenuation 2/{pi}, introduces large angular errors (mean of 52{degrees}), it frequently supported automated structure solution: in paired Phenix AutoBuild tests, 705 of 894 binary initializers met a conservative joint criterion of final Free R [≤] 30% and relative chain recovery [≥] 70%, within a 20.0-2.5 [A] resolution window. To rank candidate seeds without rebuilding, we developed a branch-balanced Basin Score from inexpensive density-modification and map-connectivity observables computed at 20.0-3.5 [A]. The empirical score quickly separates productive from unproductive initializers before AutoBuild. Controlled phase inversion shows that basin compatibility decays gradually and can reappear in an anti-phase-related branch, indicating that buildability is not confined to a single neighborhood around the reference phase set but extends to a much broader field. These results recast phase initialization as basin entry and support future symmetry-aware, binary phase-search strategies.

Bovine ephemeral fever virus (BEFV), a member of the Rhabdoviridae family, is an arthropod-borne pathogen that causes acute febrile disease in cattle. The structural basis of its genome encapsidation and virion assembly remains unexplored, with the current knowledge largely limited to predictions derived from bioinformatic comparisons with other rhabdoviruses. Furthermore, the structural principles that permit the formation of variable-diameter nucleocapsids resulting in the distinctive bullet-shaped morphology of rhabdoviruses remain poorly understood. Here, we report the cryo-electron microscopy structure of the BEFV nucleoprotein (N) in complex with RNA, in the absence of other viral components. The complex predominantly forms circular decameric oligomers that we propose to act as nucleation intermediates during assembly of the bullet-shaped nucleocapsids. Direct subunit interactions are limited to a small polar surface area, with additional intersubunit links mediated by flexible N- and C-terminal loops. These interfaces generate a structurally plastic oligomeric lattice in which neighbouring N subunits can undergo substantial rigid-body rotations and positional rearrangements while preserving conserved local contacts and continuous RNA encapsidation. Such quasi-equivalent interactions provide a plausible mechanism for accommodating the progressive changes in helical diameter required for the transition from the highly curved bullet tip to the wider cylindrical trunk of rhabdovirus nucleocapsids. The assembly is stabilised by the bound RNA molecule, where nine RNA bases are accommodated by each N subunit. The RNA-binding mechanism is consistent with that of VSV, the closest BEFV homologue characterised structurally, but differs at about half of the RNA-binding residues, demonstrating the versatility of the nucleoprotein scaffold in interacting with ssRNA. Comparative analysis with other rhabdoviruses, as well as negative-sense RNA viruses with constant-diameter nucleocapsids, such as Ebola, further confirms the structural features that enable bullet-shaped versus cylindrical nucleocapsid assembly.

Four-dimensional scanning transmission electron microscopy (4D-STEM) enables the acquisition of diffraction patterns at every probe position in a dense array. For imaging applications this approach offers significant benefits in terms of spatial resolution and contrast enhancement. In this work, we present the development of a synchronous scan generator integrated with SerialEM software to enable automation of complex experimental protocols such as tomography. The proposed hardware functions as an interface between SerialEM, the scan controls of the microscope, a fast annular dark-field detector, and a synchronized trigger for a pixelated detector. Our previous implementation, named SavvyScan, relied on a dedicated computer equipped with a multichannel acquisition and signal-generation cards, as well as a separate microcontroller for synchronization. Here, we report a low-cost implementation based on a Red Pitaya board, utilizing direct programming of its embedded FPGA and Linux server components. We provide detailed instructions for system installation and operation, along with practical guidance for modifying the source code. System performance is validated through oscilloscope measurements and imaging of a replica grating sample. The utility of the approach is further demonstrated by generating a 3D electron tomogram of a cryogenic sample of mitochondria from a tilt series of shadow montage projections.

In an infection, Hepatitis B Virus (HBV) core protein (HBc) normally assembles into icosahedral capsids. Capsid Assembly Modulators (CAMs) are direct acting antivirals that induce HBc mis-assembly and are the subject of active research and development. Two versions of HBc are used in structural studies of CAM-HBc complexes: Cp150 and Cp149-Y132A. Cp150 forms empty icosahedral capsids that are structurally indistinguishable from those found in virions. The Y132A mutation of Cp149 leads to an assembly defective soluble protein that crystalizes as flat hexagonal sheets, where the hexagons resemble icosahedral quasi-sixfold vertices. In this study, we compare structures of CAM-bound Cp150 to CAM-bound Cp149-Y132A. In capsids, the residues forming the CAM site shift to match the structure of bound CAMs, an induced fit. In Cp149-Y132A crystals, CAM sites show little structural adjustment in response to different CAMs binding. In turn, the array of residues that interact with CAMs varies from CAM to CAM in capsid structures but remains nearly constant in Cp149-Y132A crystals. These results illustrate important differences between CAM binding in Cp149-Y132A and Cp150 structures that will contribute to future CAM design.

Room temperature serial crystallography offers advantages over conventional cryo-crystallography, such as simplified crystal handling and the possibility to avoid potential artefacts associated with cryo-trapping. However, to be considered as an alternative for drug discovery, where compound availability may be limited and speed of structure delivery is a key factor, it suffers from several limitations. To address these challenges, we have optimized a serial crystallography workflow for ligand soaking, data collection and data processing, significantly reducing time and reagent consumption to make it a viable option for drug discovery applications, herein exemplified by crystallographic fragment screening. Our approach incorporates the use of dried-in fragment cocktails on fixed target supports, compatible with 96-well plates for crystal soaking, and an efficient data processing pipeline tailored for serial crystallography. To validate our workflow, we conducted an in-crystal fragment screen at room temperature on the protein soluble epoxide hydrolase. The screen comprised 384 compounds and resulted in identification of 40 fragment binders corresponding to a hit rate of 10.4 %. The resulting room-temperature structures are of high quality and reveal opportunities for specific interaction within the highly hydrophobic active site of soluble epoxide hydrolase. Finally, we discuss potential avenues for further workflow optimization, highlighting the future potential of this approach for drug discovery. SynopsisWe have developed a workflow that allowed us to efficiently conduct a fragment screen at room temperature using serial crystallography, of interest for future drug discovery campaigns.

Optical microscopy is fundamental to modern life-science research, yet interpreting its results requires precise modelling of point spread functions (PSFs) within complex environments. This manuscript introduces a versatile and efficient approach to wave-optical PSF calculations that extends existing frameworks by incorporating detection PSF modelling through the principle of reciprocity. Accompanying this work is a free MATLAB software package centred on a single, minimalistic core function, PlaneWaveExc.m, which utilizes a plane-wave superposition based on the Richards-Wolf model. Despite its simplicity, the framework accounts for "real-life" complexities such as systemic aberrations, arbitrary amplitude and phase modulations, and stratified media with complex-valued refractive indices. We demonstrate the softwares broad applicability through diverse case studies, including single-molecule imaging, STED microscopy, the segmented aperture of the James Webb Space Telescope, and coherent wide-field iSCAT microscopy. Each example is supported by dedicated scripts to facilitate adaptation for specific research needs.

Double-stranded DNA minicircles have been observed in a variety of biological settings and are also widely employed in biotechnology, therapeutic applications, and basic research. Here, we report a cryo-EM structure of a 95-basepair minicircle (dsMC95) at a 5.3 [A] resolution. dsMC95 forms a closed ring as designed and no local deformation is observed. The two DNA strands are fully resolved, with the major and minor grooves clearly distinguishable. Analysis reveals a nine-fold periodicity in the helical twist, which corresponds to approximately 10.56 base pairs per turn. Together with groove width analysis, the data indicate that dsMC95 maintains a B-DNA configuration. The dsMC95 ring exhibits an in-plane ellipticity of 1.13 and an out-of-plane displacement of 15{degrees}, with differences in out-of-plane displacements observed between the two half-segments. The dsMC95 structure, which is the only free DNA cryo-EM structure with a resolution better than 6 [A] to date, allows comparison to other structures to better understand DNA physical features such as bending. The findings advance our understanding of DNA structure under topological constraints and may inform studies of naturally occurring small circular DNA as well as the manipulation of DNA in nanotechnology applications.

Single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution structure determination of diverse biomolecules. Because the high vacuum required for electron microscopy prevents the imaging of liquid-phase samples, cryo-EM samples are prepared by plunging the sample into a cryogen, rapidly cooling the sample and suspending the ensemble of biomolecules in a matrix of water glass. However, the effects of this vitrification on the biomolecular ensemble are unknown, complicating efforts to use cryo-EM to derive conformational ensembles of biomolecules. To study these effects, we carried out extensive molecular dynamics simulations (over 50 milliseconds) of the Trp-cage miniprotein at equilibrium and undergoing rapid cooling. We simulated seven cooling rates spanning three orders of magnitude, with the slowest coolings matching experimental rates. By inspecting molecular mobility and density-temperature equations of state for water with and without protein, we found that water vitrification is unaltered by the protein. To track protein conformation changes, and to relate them to conformational kinetics, we made a Markov State Model (MSM) of Trp-cage from 5.4 milliseconds of equilibrium sampling at 277 K. We observed that MSM states with a characteristic time longer than the duration of the non-equilibrium cooling, tend to be more robust to artefacts induced by such cooling. Critically, although we observe that some states vanish in the equilibrium ensemble at 230 K, none do in our nonequilibrium cooled ensembles. However, to account for perturbations induced by nonequilibrium cooling for more labile states, we developed a thermodynamic inference framework for recovering equilibrium populations from the measured vitrified ensembles. These results indicate that cryo-EM has the capacity to be a reliable and accurate biophysical technique for the study of biomolecular ensembles. SignificanceCryogenic electron microscopy images biomolecules trapped in vitreous ice. To vitrify the sample, it must be cooled over the course of 22 microseconds. However, the degree to which this cooling causes the ensemble of the molecules to be perturbed from equilibrium is unknown. Here we present extensive molecular dynamics simulations to quantify the equilibrium dynamics of the Trp-cage miniprotein and the effects of cooling on its conformational ensemble. By simulating cooling at seven different rates, including the slowest experimental rates that still result in vitrification, we connect the kinetic properties of a proteins conformational state to the change in state population from cooling. We show that cooling-induced population shifts are small but observable. We further introduce a thermodynamic-inference method to recover equilibrium populations from the cooled ensembles.

The emergence of single-particle cryoEM as a powerful method for structure determination has in large part been fueled by its ability to resolve both single static structures and complex conformational landscapes. Indeed, modern approaches to the heterogeneous reconstruction task can resolve 100s-1,000s of different maps from a single cryoEM dataset. How accurate these algorithms are, however, has proven difficult to rigorously assess, due to a lack of suitable benchmark datasets containing both realistic noise features and ground-truth labels. To address this obstacle, we recently developed a series of benchmark datasets that leverage the targeting power of Cas9 and the programmable heterogeneity of DNA to newly offer access to ground-truth per-particle structural labels in real data. Here, we challenged two popular heterogeneous reconstruction algorithms with mixed particle stacks resampled in silico from these datasets, finding that existing approaches resolve the encoded heterogeneity with limited accuracy. In particular, in realistic particle stacks with complex, multi-scale, and multi-axis heterogeneity, we observed that reconstruction of encoded heterogeneity depended strongly on the application of prior information about where heterogeneity was expected, and that individual particle assignments were made with significant error even when the correct structural states were reconstructed. Both molecular breathing motions and data collection features, such as defocus and projection angle, contributed to the observed particle assignment error. These results highlight important shortcomings of existing heterogeneous reconstruction methods and suggest new avenues for method development in both data collection strategies and in heterogeneous classification and reconstruction algorithms.