RESUMEN
The hippocampus creates a cognitive map of the external environment by encoding spatial and self-motion-related information. However, it is unclear whether hippocampal neurons could also incorporate internal cognitive states reflecting an animal's exploratory intention, which is not driven by rewards or unexpected sensory stimuli. In this study, a subgroup of CA1 neurons was found to encode both spatial information and animals' investigatory intentions in male mice. These neurons became active before the initiation of exploration behaviors at specific locations and were nearly silent when the same fields were traversed without exploration. Interestingly, this neuronal activity could not be explained by object features, rewards, or mismatches in environmental cues. Inhibition of the lateral entorhinal cortex decreased the activity of these cells during exploration. Our findings demonstrate that hippocampal neurons may bridge external and internal signals, indicating a potential connection between spatial representation and intentional states in the construction of internal navigation systems.
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Intención , Navegación Espacial , Masculino , Ratones , Animales , Percepción Espacial/fisiología , Hipocampo/fisiología , Corteza Entorrinal , Señales (Psicología) , Navegación Espacial/fisiologíaRESUMEN
Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.
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Péptidos , Receptor de Melanocortina Tipo 4 , Humanos , Péptidos/farmacología , Ligandos , Diseño de Fármacos , Receptor de Melanocortina Tipo 3 , Receptores de MelanocortinaRESUMEN
Actin mediates insulin secretion in pancreatic ß-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E ß-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a "netlike" to a "blooming" architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.
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Actinas , Células Secretoras de Insulina , Secreción de Insulina , Actinas/metabolismo , Glucosa/metabolismo , Tomografía con Microscopio Electrónico , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Citoesqueleto de Actina/metabolismoRESUMEN
Despite advances in characterizing the structures and functions of G protein-coupled receptors (GPCRs), our understanding of GPCR activation and signaling is still limited by the lack of information on conformational dynamics. It is particularly challenging to study the dynamics of GPCR complexes with their signaling partners because of their transient nature and low stability. Here, by combining cross-linking mass spectrometry (CLMS) with integrative structure modeling, we map the conformational ensemble of an activated GPCR-G protein complex at near-atomic resolution. The integrative structures describe heterogeneous conformations for a high number of potential alternative active states of the GLP-1 receptor-Gs complex. These structures show marked differences from the previously determined cryo-EM structure, especially at the receptor-Gs interface and in the interior of the Gs heterotrimer. Alanine-scanning mutagenesis coupled with pharmacological assays validates the functional significance of 24 interface residue contacts only observed in the integrative structures, yet absent in the cryo-EM structure. Through the integration of spatial connectivity data from CLMS with structure modeling, our study provides a new approach that is generalizable to characterizing the conformational dynamics of GPCR signaling complexes.
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The recently developed double-click reaction sequence [G. Meng et al., Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.
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Receptor del Péptido 1 Similar al Glucagón , Péptidos , Regulación Alostérica , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Péptidos/química , Triazoles/químicaRESUMEN
The hydroxycarboxylic acid receptor 2 (HCA2) agonist niacin has been used as treatment for dyslipidemia for several decades albeit with skin flushing as a common side-effect in treated individuals. Extensive efforts have been made to identify HCA2 targeting lipid lowering agents with fewer adverse effects, despite little being known about the molecular basis of HCA2 mediated signalling. Here, we report the cryo-electron microscopy structure of the HCA2-Gi signalling complex with the potent agonist MK-6892, along with crystal structures of HCA2 in inactive state. These structures, together with comprehensive pharmacological analysis, reveal the ligand binding mode and activation and signalling mechanisms of HCA2. This study elucidates the structural determinants essential for HCA2 mediated signalling and provides insights into ligand discovery for HCA2 and related receptors.
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Niacina , Humanos , Niacina/farmacología , Ligandos , Microscopía por Crioelectrón , Transducción de Señal , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Glucagon-like peptide-1 receptor (GLP-1R) is a critical therapeutic target for type 2 diabetes mellitus (T2DM). The GLP-1R cellular signaling mechanism relevant to insulin secretion and blood glucose regulation has been extensively studied. Numerous drugs targeting GLP-1R have entered clinical treatment. However, novel functional molecules with reduced side effects and enhanced therapeutic efficacy are still in high demand. In this review, we summarize the basis of GLP-1R cellular signaling, and how it is involved in the treatment of T2DM. We review the functional molecules of incretin therapy in various stages of clinical trials. We also outline the current strategies and emerging techniques that are furthering the development of novel therapeutic drugs for T2DM and other metabolic diseases.
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Diabetes Mellitus Tipo 2 , Incretinas , Humanos , Incretinas/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Insulina/metabolismo , Transducción de SeñalRESUMEN
Investigating the 3D structures and rearrangements of organelles within a single cell is critical for better characterizing cellular function. Imaging approaches such as soft X-ray tomography have been widely applied to reveal a complex subcellular organization involving multiple inter-organelle interactions. However, 3D segmentation of organelle instances has been challenging despite its importance in organelle characterization. Here we propose an intensity-based post-processing tool to identify and separate organelle instances. Our tool separates sphere-like (insulin vesicle) and columnar-shaped organelle instances (mitochondrion) based on the intensity of raw tomograms, semantic segmentation masks, and organelle morphology. We validate our tool using synthetic tomograms of organelles and experimental tomograms of pancreatic ß-cells to separate insulin vesicle and mitochondria instances. As compared to the commonly used connected regions labeling, watershed, and watershed + Gaussian filter methods, our tool results in improved accuracy in identifying organelles in the synthetic tomograms and an improved description of organelle structures in ß-cell tomograms. In addition, under different experimental treatment conditions, significant changes in volumes and intensities of both insulin vesicle and mitochondrion are observed in our instance results, revealing their potential roles in maintaining normal ß-cell function. Our tool is expected to be applicable for improving the instance segmentation of other images obtained from different cell types using multiple imaging modalities.
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Imagenología Tridimensional , Insulinas , Imagenología Tridimensional/métodos , Orgánulos/química , Tomografía , Rayos XRESUMEN
Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson's disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds.
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Nucleósidos , Receptor de Adenosina A2A , Antagonistas del Receptor de Adenosina A2/química , Antagonistas del Receptor de Adenosina A2/farmacología , Cristalografía por Rayos X , Conformación Molecular , Receptor de Adenosina A2A/química , TiofenosRESUMEN
Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.
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Células Secretoras de Insulina , Insulina , Vesículas Secretoras , Zinc , Animales , Glucemia , Línea Celular , Insulina/análisis , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestructura , Ratas , Vesículas Secretoras/química , Vesículas Secretoras/metabolismo , Espectrometría por Rayos X , Difracción de Rayos X , Zinc/análisisRESUMEN
The technique of cryogenic-electron microscopy (cryo-EM) has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species. This report describes the structural characterization of a fully active human apelin receptor (APJR) complexed with heterotrimeric G protein observed in both 2:1 and 1:1 stoichiometric ratios. We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation. Protein preparations, in the presence of the endogenous peptide ligand ELA or a synthetic small molecule, both demonstrate these mixed stoichiometric states. Structural differences in G protein engagement between dimeric and monomeric APJR suggest a role for the stoichiometry of G protein-coupled receptor- (GPCR-)G protein coupling on downstream signaling and receptor pharmacology. Furthermore, a small, hydrophobic dimer interface provides a starting framework for additional class A GPCR dimerization studies. Together, these findings uncover a mechanism of versatile regulation through oligomerization by which GPCRs can modulate their signaling.
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Proteínas de Unión al GTP , Receptores Acoplados a Proteínas G , Receptores de Apelina/química , Receptores de Apelina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Receptores Acoplados a Proteínas G/química , Transducción de SeñalRESUMEN
BACKGROUND: In drug-resistant epilepsy, surgical resection of the epileptic focus can end seizures. However, success is dependent on the ability to identify foci locations and, unfortunately, current methods like electrophysiology and positron emission tomography can give contradictory results. During seizures, glucose is metabolized at epileptic foci through aerobic glycolysis, which can be imaged through the oxygen-glucose index (OGI) biomarker. However, inter-ictal (between seizures) OGI changes have not been studied, which has limited its application. METHODS: 18 healthy controls and 24 inter-ictal, temporal lobe epilepsy patients underwent simultaneous positron emission tomography (PET) and magnetic resonance imaging (MRI) scans. We used [18F]fluorodeoxyglucose-PET (FDG-PET) to detect cerebral glucose metabolism, and calibrated functional MRI to acquire relative oxygen consumption. With these data, we calculated relative OGI maps. FINDINGS: While bilaterally symmetrical in healthy controls, we observed, in patients during the inter-ictal period, higher OGI ipsilateral to the epileptic focus than contralateral. While traditional FDG-PET results and temporal lobe OGI results usually both agreed with invasive electrophysiology, in cases where FDG-PET disagreed with electrophysiology, temporal lobe OGI agreed with electrophysiology, and vice-versa. INTERPRETATION: As either our novel epilepsy biomarker or traditional approaches located foci in every case, our work provides promising insights into metabolic changes in epilepsy. Our method allows single-session OGI measurement which can be useful in other diseases. FUNDING: This work was supported by ShanghaiTech University, the Shanghai Municipal Government, the National Natural Science Foundation of China Grant (No. 81950410637) and Shanghai Municipal Key Clinical Specialty (No. shslczdzk03403). F. H. and P. H. were supported by USA National Institute of Health grants (R01 NS-100106, R01 MH-067528).Z. W. was supported by the Key-Area Research and Development Program of Guangdong Province (2019B030335001), National Natural Science Foundation of China (No. 82151303), and National Key R&D Program of China (No. 2021ZD0204002).
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Epilepsia , Fluorodesoxiglucosa F18 , Biomarcadores/metabolismo , China , Electroencefalografía , Epilepsia/metabolismo , Glucosa/metabolismo , Glucólisis , Humanos , Imagen por Resonancia Magnética , Tomografía de Emisión de Positrones/métodos , Convulsiones/diagnóstico por imagen , Tomografía Computarizada por Rayos XRESUMEN
The mesoscale description of the subcellular organization informs about cellular mechanisms in disease state. However, applications of soft X-ray tomography (SXT), an important approach for characterizing organelle organization, are limited by labor-intensive manual segmentation. Here we report a pipeline for automated segmentation and systematic analysis of SXT tomograms. Our approach combines semantic and first-applied instance segmentation to produce separate organelle masks with high Dice and Recall indexes, followed by analysis of organelle localization based on the radial distribution function. We demonstrated this technique by investigating the organization of INS-1E pancreatic ß-cell organization under different treatments at multiple time points. Consistent with a previous analysis of a similar dataset, our results revealed the impact of glucose stimulation on the localization and molecular density of insulin vesicles and mitochondria. This pipeline can be extended to SXT tomograms of any cell type to shed light on the subcellular rearrangements under different drug treatments.
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Células Secretoras de Insulina , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismoRESUMEN
As part of a project to build a spatiotemporal model of the pancreatic ß-cell, we are creating an immersive experience called "World in a Cell" that can be used to integrate and create new educational tools. To do this, we have developed a new visual design language that uses tetrahedral building blocks to express the structural features of biological molecules and organelles in crowded cellular environments. The tetrahedral language enables more efficient animation and user interaction in an immersive environment.
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LenguajeRESUMEN
Since their discovery, nicotinic acetylcholine receptors (nAChRs) have been extensively studied to understand their function, as well as the consequence of alterations leading to disease states. Importantly, these receptors represent pharmacological targets to treat a number of neurological and neurodegenerative disorders. Nevertheless, their therapeutic value has been limited by the absence of high-resolution structures that allow for the design of more specific and effective drugs. This article offers a comprehensive review of five decades of research pursuing high-resolution structures of nAChRs. We provide a historical perspective, from initial structural studies to the most recent X-ray and cryogenic electron microscopy (Cryo-EM) nAChR structures. We also discuss the most relevant structural features that emerged from these studies, as well as perspectives in the field.
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Enfermedades del Sistema Nervioso/metabolismo , Receptores Nicotínicos/química , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Terapia Molecular Dirigida , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Conformación Proteica , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismoRESUMEN
Azobenzene-embedded photoswitchable ligands are the widely used chemical tools in photopharmacological studies. Current approaches to azobenzene introduction rely mainly on the isosteric replacement of typical azologable groups. However, atypical scaffolds may offer more opportunities for photoswitch remodeling, which are chemically in an overwhelming majority. Herein, we investigate the rational remodeling of atypical scaffolds for azobenzene introduction, as exemplified in the development of photoswitchable ligands for the cannabinoid receptor 2 (CB2). Based on the analysis of residue-type clusters surrounding the binding pocket, we conclude that among the three representative atypical arms of the CB2 antagonist, AM10257, the adamantyl arm is the most appropriate for azobenzene remodeling. The optimizing spacer length and attachment position revealed AzoLig 9 with excellent thermal bistability, decent photopharmacological switchability between its two configurations, and high subtype selectivity. This structure-guided approach gave new impetus in the extension of new chemical spaces for tool customization for increasingly diversified photo-pharmacological studies and beyond.
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Compuestos Azo/farmacología , Receptor Cannabinoide CB2/metabolismo , Animales , Compuestos Azo/síntesis química , Compuestos Azo/metabolismo , Compuestos Azo/efectos de la radiación , Células CHO , Cricetulus , Diseño de Fármacos , Humanos , Ligandos , Luz , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Receptor Cannabinoide CB2/químicaRESUMEN
Comprehensive modeling of a whole cell requires an integration of vast amounts of information on various aspects of the cell and its parts. To divide and conquer this task, we introduce Bayesian metamodeling, a general approach to modeling complex systems by integrating a collection of heterogeneous input models. Each input model can in principle be based on any type of data and can describe a different aspect of the modeled system using any mathematical representation, scale, and level of granularity. These input models are 1) converted to a standardized statistical representation relying on probabilistic graphical models, 2) coupled by modeling their mutual relations with the physical world, and 3) finally harmonized with respect to each other. To illustrate Bayesian metamodeling, we provide a proof-of-principle metamodel of glucose-stimulated insulin secretion by human pancreatic ß-cells. The input models include a coarse-grained spatiotemporal simulation of insulin vesicle trafficking, docking, and exocytosis; a molecular network model of glucose-stimulated insulin secretion signaling; a network model of insulin metabolism; a structural model of glucagon-like peptide-1 receptor activation; a linear model of a pancreatic cell population; and ordinary differential equations for systemic postprandial insulin response. Metamodeling benefits from decentralized computing, while often producing a more accurate, precise, and complete model that contextualizes input models as well as resolves conflicting information. We anticipate Bayesian metamodeling will facilitate collaborative science by providing a framework for sharing expertise, resources, data, and models, as exemplified by the Pancreatic ß-Cell Consortium.
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Modelos Biológicos , Teorema de Bayes , Simulación por Computador , Humanos , Modelos LinealesRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that exerts crucial metabolic functions by binding and activating its cognate receptor, GIPR. As an important therapeutic target, GIPR has been subjected to intensive structural studies without success. Here, we report the cryo-EM structure of the human GIPR in complex with GIP and a Gs heterotrimer at a global resolution of 2.9 Å. GIP adopts a single straight helix with its N terminus dipped into the receptor transmembrane domain (TMD), while the C terminus is closely associated with the extracellular domain and extracellular loop 1. GIPR employs conserved residues in the lower half of the TMD pocket to recognize the common segments shared by GIP homologous peptides, while uses non-conserved residues in the upper half of the TMD pocket to interact with residues specific for GIP. These results provide a structural framework of hormone recognition and GIPR activation.
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Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/metabolismo , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/metabolismo , Animales , Línea Celular , Microscopía por Crioelectrón , Humanos , Ligandos , Simulación de Dinámica Molecular , Conformación Proteica , Conformación Proteica en Hélice alfa , Dominios ProteicosRESUMEN
Type 2 diabetes is characterized by ß and α cell dysfunction. We used phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) to monitor oxidative phosphorylation and glycolysis in living islet cells before and after glucose stimulation. In healthy cells, glucose enhanced oxidative phosphorylation in ß cells and suppressed oxidative phosphorylation in α cells. In Type 2 diabetes, glucose increased glycolysis in ß cells, and only partially suppressed oxidative phosphorylation in α cells. FLIM uncovers key perturbations in glucose induced metabolism in living islet cells and provides a sensitive tool for drug discovery in diabetes.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucosa/farmacología , Células Secretoras de Insulina/metabolismo , Imagen Molecular/métodos , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/patología , Células Secretoras de Glucagón/efectos de los fármacos , Glucólisis , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Masculino , Ratones , Microscopía Fluorescente , Fosforilación Oxidativa , Ratas , Ratas Sprague-Dawley , Ratas TransgénicasRESUMEN
Cryo-electron tomography provides the opportunity for unsupervised discovery of endogenous complexes in situ. This process usually requires particle picking, clustering and alignment of subtomograms to produce an average structure of the complex. When applied to heterogeneous samples, template-free clustering and alignment of subtomograms can potentially lead to the discovery of structures for unknown endogenous complexes. However, such methods require scoring functions to measure and accurately rank the quality of aligned subtomogram clusters, which can be compromised by contaminations from misclassified complexes and alignment errors. Here, we provide the first study to assess the effectiveness of more than 15 scoring functions for evaluating the quality of subtomogram clusters, which differ in the amount of structural misalignments and contaminations due to misclassified complexes. We assessed both experimental and simulated subtomograms as ground truth data sets. Our analysis showed that the robustness of scoring functions varies largely. Most scores were sensitive to the signal-to-noise ratio of subtomograms and often required Gaussian filtering as preprocessing for improved performance. Two scoring functions, Spectral SNR-based Fourier Shell Correlation and Pearson Correlation in the Fourier domain with missing wedge correction, showed a robust ranking of subtomogram clusters without any preprocessing and irrespective of SNR levels of subtomograms. Of these two scoring functions, Spectral SNR-based Fourier Shell Correlation was fastest to compute and is a better choice for handling large numbers of subtomograms. Our results provide a guidance for choosing an accurate scoring function for template-free approaches to detect complexes from heterogeneous samples.