Data associated with the submitted manuscript "Resolving Functional States in Cryo-EM Datasets with JANAS"

Here we include datasets associated with the application of JANAS (Joint ANAlysis of Stacks), a cryo-EM data processing pipeline for particle selection and 3D class reassignment based on the Structural Cross-correlation Index (SCI). The deposited data include: EMPIAR-10308 (5-HT1BR–Go receptor complex), used to assess SCI-based global particle selection and a focused analysis of the ligand-binding pocket; EMPIAR-10667 (46Q HTT–HAP40 complex), used to evaluate class reassignment on the deposited stack and reproducibility on an independently repicked stack; a newly acquired O. iheyensis group II intron dataset; the OLE RNA homodimer (EMPIAR-12707), used as an RNA-only benchmark; and an SCI_test archive providing the source data for the SCI characterisation and method-comparison experiments. Raw particle stacks from EMPIAR-10308, EMPIAR-10667 and EMPIAR-12707 are already publicly deposited and thus not included here. For the group II intron dataset, particle stacks for J317 and J246 are provided. Datasets also include the refined 3D maps, atomic models and related metadata. EMPIAR_10308_particle_selection.zip This archive contains the SCI-based particle selection results applied to EMPIAR-10308 (5-HT1BR–Go receptor complex bound to donitriptan). It also includes half-maps, ModelAngelo automatic models obtained from the unprocessed maps, and the *_stackNotationEMPIAR.star files for particle stacks corresponding to the subset of particles ranked and selected. Directories include deposited_data, corresponding to the original EMPIAR-10308 deposit; selected_244565_particles, with the selected subset; and selected_244565_particles_refined, with the angular-refined maps reconstructed from the selected particles. EMPIAR_10308_ligand.zip This archive includes focused classification and class-reassignment data for the ligand-binding region of the 5-HT1BR–Go receptor complex from EMPIAR-10308. Files include particle stacks, class assignments and refined maps corresponding to the five final classes identified in Fig. S4 of the manuscript, in support of a comparative evaluation of conformational states within the binding pocket, and to the two selected classes carried forward for ligand modelling: the Ser334-proximal pose (16,736 particles) and the linker-reoriented pose (15,349 particles). The archive contains refined maps (half-maps and post-processed maps), local-resolution estimations, masks, the corresponding .star files of particles contributing to each class, the cryoSPARC reference reconstructions used as input, the signal-subtracted reconstructions, and the two new atomic models of the ligand-binding pocket (Ser334.pdb, Linker-reoriented.pdb) together with the deposited reference model (PDB 6G79). EMPIAR_10667_final_classes_selected.zip and EMPIAR_10667_3dFSC_analysis.zip These archives contain the class reassignment and 3D refinement results for EMPIAR-10667 (46Q HTT–HAP40 complex). Particular focus is given to flexible regions such as the N-terminal BΦ motif, and domain-resolved refinements are available for further comparative analysis. Included files are final refined maps for each selected class, ModelAngelo traces for each selected class, .star files of selected particles, local resolution maps, directional 3D FSC volumes and correlation plots, and atomic models corresponding to each class, including mask and map-fitting metadata. EMPIAR_10667_repick.zip This archive contains the SCI-seeded repicking and class-reassignment results for EMPIAR-10667, testing the reproducibility of the BΦ-motif conformations through an independent upstream coordinate-generation route (Fig. S7 of the manuscript). A small SCI-scored, orientation-balanced subset was used to seed cryoSPARC template matching and Topaz repicking from the original micrographs, yielding a stack of 458,445 particles with reduced preferred orientation, which was then processed by non-uniform refinement, 3D class reassignment and per-class selection. Included files are the seeding subset, the topaz-repicked metadata and associated cryoSPARC reconstruction, the masks used for reassignment, the per-class reassignment outputs, and the per-class selected reconstructions with half-maps, local-resolution maps and atomic models, together with the reassignment and selection scripts. groupII_intron.zip This dataset includes newly acquired cryo-EM data for the Oceanobacillus iheyensis group II intron, with the aim of facilitating detailed exploration of conformational heterogeneity in the catalytic core and peripheral regions. The archive contains particle stacks for J317 and J246. Further, it contains the seven refined 3D classes and associated data: metadata .star files of particles assigned to each class, full and half maps for all classes, masks, local resolution maps and FSC plots, validation reports for deposited maps on the PDB, a data processing summary, and comparative resolution data. EMPIAR_12707.zip This archive contains the SCI-based particle-selection results for the OLE RNA homodimer (EMPIAR-12707; deposited map EMD-48163; deposited model PDB 9MCW), a 373 kDa RNA-only assembly used as an additional benchmark with a stable central scaffold and local-resolution variation in peripheral regions associated with the proposed OapA/OapC binding architecture (Fig. 7 of the manuscript). Three iterative rounds of particle selection with adaptive masking and angular refinement between rounds retained 64,481 of the initial 87,716 particles. Included files are: the particle metadata (.star files) for the full deposited stack and for the selected subset; the reconstruction and half-maps of the selected subset; reconstructions of the major 3D classes obtained from the cumulatively excluded particles (supporting Fig. S12); the adaptive mask and ChimeraX session; and the atomic model refined against the selected reconstruction starting from PDB 9MCW. SCI_test.zip This archive provides the source data and supporting files for the SCI characterisation and method-comparison experiments. It supports the SCI vs LoG sensitivity test on a controlled image with known geometric perturbations (Fig. S1B), the defocus-resampling control on EMPIAR-10308 (Fig. S2), and the rank-correlation analysis between SCI and CC, NMI, SSIM and MVPD on EMPIAR-10308 and EMPIAR-10667 (Supplementary Results 4.1). Included files are: the rotated reference images and processing script for the LoG sensitivity test; the per-method 10,000-particle subsets used for the defocus-resampling control; the Euler-balanced normalised rank .star files for each metric on both datasets, together with the reconstructions and masks used for ranking; and the scripts for computing the per-dataset Spearman ρ and Kendall τ rank-correlation values reported in the manuscript.本数据集关联EMPROVE的应用，EMPROVE是一款用于结构生物学中粒子挑选与三维类别重分配的冷冻电镜（cryo-EM）数据处理流程。本次提交的数据包括：用于评估基于EMPROVE的粒子挑选效果的EMPIAR-10308数据集，其样本为结合了多尼曲普坦的5-羟色胺5-HT1BR-Go受体复合物；针对该数据集配体结合口袋的聚焦分析；用于评估取向偏差校正与构象分辨率的EMPIAR-10667数据集，其样本为46Q HTT-HAP40复合物；以及本次新采集的、使用EMPROVE处理以用于RNA结构分析的II组内含子数据集。EMPIAR-10308与EMPIAR-10667的原始粒子栈已公开提交（因此未包含在本归档中），本次仅提交基于EMPROVE的上述数据集的粒子挑选结果。针对II组内含子数据集，提供了J317与J246的粒子栈。本数据集还包含经EMPROVE精修的三维密度图及相关元数据。 EMPIAR_10308_EMPROVE_particle_selection.zip 该归档包含针对EMPIAR-10308（结合了多尼曲普坦的5-HT1BR-Go受体复合物）的基于EMPROVE的粒子挑选结果。此外还包含半密度图、从未经处理的密度图中获取的ModelAngelo自动模型，以及对应经EMPROVE排名筛选的粒子子集的"*_stackNotationEMPIAR.star"文件。目录结构包括：deposited_data，对应原始EMPIAR-10308提交数据；selected_244565_particles，存放EMPROVE筛选得到的粒子子集；selected_244565_particles_refined，存放由筛选粒子重构得到的经角度精修的密度图。 EMPIAR_10308_ligand_classReassignment.zip 该归档包含针对EMPIAR-10308中5-HT1BR-Go受体复合物配体结合区域的聚焦分类与重分配数据。文件包括粒子栈、类别分配结果，以及对应论文图S3中鉴定的5个最终类别的精修密度图，用于辅助评估结合口袋内的构象状态差异。归档内含精修密度图（半密度图与后处理密度图）、局部分辨率估计结果、对应每个类别的粒子的.star文件，以及与配体相关的新PDB模型。 EMPIAR_10667_final_classes_selected.zip 与 EMPIAR_10667_3dFSC_analysis.zip 这两个归档包含针对EMPIAR-10667（46Q HTT-HAP40复合物）的基于EMPROVE的类别重分配与三维精修结果。重点关注N端BΦ基序等柔性区域，同时提供域解析精修结果以支持进一步的比较分析。包含的文件有：每个筛选类别的最终精修密度图、每个类别的ModelAngelo模型、筛选粒子的.star文件、局部分辨率图、定向三维FSC体积与相关性图、对应每个类别的原子模型（包含掩膜与密度图拟合元数据）。 groupII_intron.zip 本数据集包含新采集的海洋嗜热杆菌（Oceanobacillus iheyensis）II组内含子冷冻电镜数据，经EMPROVE处理，旨在助力催化核心与外围区域构象异质性的详细研究。归档包含J317与J246的粒子栈，此外还包含7个经EMPROVE精修的三维类别及相关数据：分配至每个类别的粒子的元数据.star文件、所有类别的全密度图与半密度图、掩膜、局部分辨率图与FSC曲线图、提交至PDB的密度图验证报告、数据处理总结、与标准分类方法的对比分辨率数据。