RESUMEN
Integral membrane proteins (IMPs) play central roles in cellular physiology and represent the majority of known drug targets. Single-molecule fluorescence and fluorescence resonance energy transfer (FRET) methods have recently emerged as valuable tools for investigating structure-function relationships in IMPs. This review focuses on the practical foundations required for examining polytopic IMP function using single-molecule FRET (smFRET) and provides an overview of the technical and conceptual frameworks emerging from this area of investigation. In this context, we highlight the utility of smFRET methods to reveal transient conformational states critical to IMP function and the use of smFRET data to guide structural and drug mechanism-of-action investigations. We also identify frontiers where progress is likely to be paramount to advancing the field.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Proteínas de la Membrana , Imagen Individual de Molécula , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Transferencia Resonante de Energía de Fluorescencia/métodos , Imagen Individual de Molécula/métodos , Humanos , AnimalesRESUMEN
Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to expand the chemical space available to biological therapeutics and materials, but existing technologies are still limiting. Addressing these limitations requires a deeper understanding of the mechanism of protein synthesis and how it is perturbed by nnAAs. Here we examine the impact of nnAAs on the formation and ribosome utilization of the central elongation substrate: the ternary complex of native, aminoacylated tRNA, thermally unstable elongation factor, and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer measurements, we reveal that both the (R)- and (S)-ß2 isomers of phenylalanine (Phe) disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-ß3-Phe reduce ternary complex stability by 1 order of magnitude. Consistent with these findings, (R)- and (S)-ß2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-ß3-Phe stereoisomers were utilized inefficiently. (R)-ß3-Phe but not (S)-ß3-Phe also exhibited order of magnitude defects in the rate of translocation after mRNA decoding. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include the consideration of the efficiency and stability of ternary complex formation.
RESUMEN
Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Fotones , Colorantes Fluorescentes/química , Imagen Individual de Molécula/métodosRESUMEN
Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to vastly expand the chemical space available to biological therapeutics and materials. Existing technologies limit the identity and number of nnAAs than can be incorporated into a given protein. Addressing these bottlenecks requires deeper understanding of the mechanism of messenger RNA (mRNA) templated protein synthesis and how this mechanism is perturbed by nnAAs. Here we examine the impact of both monomer backbone and side chain on formation and ribosome-utilization of the central protein synthesis substate: the ternary complex of native, aminoacylated transfer RNA (aa-tRNA), thermally unstable elongation factor (EF-Tu), and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer (FRET) measurements, we reveal the dramatic effect of monomer backbone on ternary complex formation and protein synthesis. Both the (R) and (S)-ß2 isomers of Phe disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-ß3-Phe reduce ternary complex stability by approximately one order of magnitude. Consistent with these findings, (R)- and (S)-ß2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-ß3-Phe stereoisomers were utilized inefficiently. The reduced affinities of both species for EF-Tu ostensibly bypassed the proofreading stage of mRNA decoding. (R)-ß3-Phe but not (S)-ß3-Phe also exhibited order of magnitude defects in the rate of substrate translocation after mRNA decoding, in line with defects in peptide bond formation that have been observed for D-α-Phe. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include consideration of the efficiency and stability of ternary complex formation.
RESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor.
Asunto(s)
Adenosina Trifosfato , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Tiazolidinas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Dimerización , BenzoatosRESUMEN
Accurate protein synthesis is determined by the two-subunit ribosome's capacity to selectively incorporate cognate aminoacyl-tRNA for each mRNA codon. The molecular basis of tRNA selection accuracy, and how fidelity can be affected by antibiotics, remains incompletely understood. Using molecular simulations, we find that cognate and near-cognate tRNAs delivered to the ribosome by Elongation Factor Tu (EF-Tu) can follow divergent pathways of motion into the ribosome during both initial selection and proofreading. Consequently, cognate aa-tRNAs follow pathways aligned with the catalytic GTPase and peptidyltransferase centers of the large subunit, while near-cognate aa-tRNAs follow pathways that are misaligned. These findings suggest that differences in mRNA codon-tRNA anticodon interactions within the small subunit decoding center, where codon-anticodon interactions occur, are geometrically amplified over distance, as a result of this site's physical separation from the large ribosomal subunit catalytic centers. These insights posit that the physical size of both tRNA and ribosome are key determinants of the tRNA selection fidelity mechanism.
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Magnoliopsida , Aminoacil-ARN de Transferencia , Aminoacil-ARN de Transferencia/genética , ARN Mensajero/genética , Anticodón , Ribosomas , Biosíntesis de ProteínasRESUMEN
In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems1. Although key features are conserved across evolution2, eukaryotes achieve higher-fidelity mRNA decoding than bacteria3. In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment4-6. Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.
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Bacterias , Biosíntesis de Proteínas , Humanos , Bacterias/genética , Bacterias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Imagen Individual de Molécula , Microscopía por Crioelectrón , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes1. Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure2,3. Electrophysiological properties of CFTR have been analysed for decades4-6. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Cloruros/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Conductividad Eléctrica , Electrofisiología , Transferencia Resonante de Energía de Fluorescencia , Activación del Canal Iónico , Multimerización de Proteína/genéticaRESUMEN
Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.
Asunto(s)
Productos Biológicos , Aminoacil-ARN de Transferencia , Animales , Humanos , Productos Biológicos/metabolismo , Codón/metabolismo , Guanosina Trifosfato/metabolismo , Mamíferos/genética , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Ribosomas/metabolismo , Aminoacil-ARN de Transferencia/metabolismoRESUMEN
Ternatin-family cyclic peptides inhibit protein synthesis by targeting the eukaryotic elongation factor-1α. A potentially related cytotoxic natural product ('A3') was isolated from Aspergillus, but only 4 of its 11 stereocentres could be assigned. Here, we synthesized SR-A3 and SS-A3-two out of 128 possible A3 epimers-and discovered that synthetic SR-A3 is indistinguishable from naturally derived A3. Relative to SS-A3, SR-A3 exhibits an enhanced residence time and rebinding kinetics, as revealed by single-molecule fluorescence imaging of elongation reactions catalysed by eukaryotic elongation factor-1α in vitro. An increased residence time-stereospecifically conferred by the unique ß-hydroxyl in SR-A3-was also observed in cells. Consistent with its prolonged duration of action, thrice-weekly dosing with SR-A3 led to a reduced tumour burden and increased survival in an aggressive Myc-driven mouse lymphoma model. Our results demonstrate the potential of SR-A3 as a cancer therapeutic and exemplify an evolutionary mechanism for enhancing cyclic peptide binding kinetics via stereospecific side-chain hydroxylation.
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Antineoplásicos , Imagen Individual de Molécula , Animales , Ratones , Cinética , Antineoplásicos/farmacología , Péptidos Cíclicos/farmacologíaRESUMEN
Translocation of messenger RNA (mRNA) and transfer RNA (tRNA) substrates through the ribosome during protein synthesis, an exemplar of directional molecular movement in biology, entails a complex interplay of conformational, compositional, and chemical changes. The molecular determinants of early translocation steps have been investigated rigorously. However, the elements enabling the ribosome to complete translocation and reset for subsequent protein synthesis reactions remain poorly understood. Here, we have combined molecular simulations with single-molecule fluorescence resonance energy transfer imaging to gain insights into the rate-limiting events of the translocation mechanism. We find that diffusive motions of the ribosomal small subunit head domain to hyper-swivelled positions, governed by universally conserved rRNA, can maneuver the mRNA and tRNAs to their fully translocated positions. Subsequent engagement of peptidyl-tRNA and disengagement of deacyl-tRNA from mRNA, within their respective small subunit binding sites, facilitate the ribosome resetting mechanism after translocation has occurred to enable protein synthesis to resume.
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Factor G de Elongación Peptídica , Ribosomas , Factor G de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/química , ARN de Transferencia/metabolismo , Ribosomas/metabolismoRESUMEN
Argyrins are a family of naturally produced octapeptides that display promising antimicrobial activity against Pseudomonas aeruginosa. Argyrin B (ArgB) has been shown to interact with an elongated form of the translation elongation factor G (EF-G), leading to the suggestion that argyrins inhibit protein synthesis by interfering with EF-G binding to the ribosome. Here, using a combination of cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that rather than interfering with ribosome binding, ArgB rapidly and specifically binds EF-G on the ribosome to inhibit intermediate steps of the translocation mechanism. Our data support that ArgB inhibits conformational changes within EF-G after GTP hydrolysis required for translocation and factor dissociation, analogous to the mechanism of fusidic acid, a chemically distinct antibiotic that binds a different region of EF-G. These findings shed light on the mechanism of action of the argyrin-class antibiotics on protein synthesis as well as the nature and importance of rate-limiting, intramolecular conformational events within the EF-G-bound ribosome during late-steps of translocation.
Asunto(s)
Antibacterianos , Factor G de Elongación Peptídica , Antibacterianos/metabolismo , Ácido Fusídico/farmacología , Humanos , Oligopéptidos , Factor G de Elongación Peptídica/metabolismo , Ribosomas/metabolismo , Translocación GenéticaRESUMEN
ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.
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Fosfopéptidos , Receptores Acoplados a Proteínas G , Fosfopéptidos/metabolismo , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.
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Sistema de Transporte de Aminoácidos X-AG/metabolismo , Proteínas Arqueales/metabolismo , Membrana Celular/metabolismo , Medición de Intercambio de Deuterio , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG/genética , Proteínas Arqueales/genética , Transporte Biológico , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Espectrometría de Masas , Mutación , Unión Proteica , Imagen Individual de MoléculaRESUMEN
Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity-often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Indoles/uso terapéutico , Morfolinas/uso terapéutico , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Pirimidinas/uso terapéutico , Sulfonamidas/uso terapéutico , Animales , Benzotiazoles , Western Blotting , Línea Celular Tumoral , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Desnudos , Simulación de Dinámica Molecular , Naftiridinas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2-20 per second) and with a low error rate (around 10-3 to 10-5 at each step) over thousands of cycles1. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed.
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Factor G de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/genética , Ribosomas/metabolismo , Codón , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , ARN Mensajero/genéticaRESUMEN
Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population.
RESUMEN
Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.