The observed substantial loss of Arctic sea ice has raised prospects of a seasonally ice-free Arctic Ocean within the foreseeable future. In this review, we summarize our current understanding of the most likely trajectory of the Arctic sea-ice cover towards this state.∘C global warming relative to pre-industrial levels. To keep chances below 5 % that the Arctic Ocean will largely be ice free in a given year, total future CO2 emissions must remain below 500 Gt.The future trajectory of the Arctic sea-ice cover can be described through a deterministic component arising primarily from future greenhouse gas emissions, and a chaotic component arising from internal variability. The deterministic component is expected to cause a largely ice-free Arctic Ocean during summer for less than 2 The Arctic Ocean will become ice free during summer before mid-century unless greenhouse gas emissions are rapidly reduced. The observed recent loss of Arctic sea ice is probably the clearest indicator of the ongoing rapid changes in the climate state of our planet. Over the past few decades, the sea-ice-covered area of the Arctic Ocean has been reduced by about half during summer , and theWe start with a brief overview of the main sources of information that have been used to estimate the future evolution of the Arctic sea-ice cover, focusing in particular on the strengths and weaknesses of the underlying models and observations. Based on this assessment, we afterwards provide a unified understanding of the response of the sea-ice cover to changes in external forcing, internal variability, and self amplification. This unified understanding is obtained by combining observation-based studies with model-based studies, allowing us to fully profit from the individual strengths of these two sources of information. In particular, we will demonstrate how quantitative estimates of the future evolution of the Arctic sea-ice cover can most robustly be obtained by combining observed quantitative information with robust underlying relationships obtained from model simulations.As in most other scientific disciplines, we have two major sources of information to examine the loss of Arctic sea ice in the real world: observational estimates and models. As both these sources of information have certain limitations, the most robust insights into the ongoing and future evolution of Arctic sea ice are obtained from combining observations and models, ideally minimizing the impact of the limitations of either .Observational records of Arctic sea ice are the best possible estimate of the real evolution of the sea-ice cover. However, all existing observational records of Arctic sea ice have three distinct limitations, which imply that these observational records alone do not allow one to fully understand the drivers of the current ice changes or to fully characterize the future evolution of the sea-ice cover.The first limitation is related to internal variability: Because the climate system is chaotic, any given set of external boundary conditions allows for an infinite number of possible climate trajectories. However, only one of these trajectories is actually realized and can be observed. This implies that observations can never capture the richness of possible trajectories that the climate system could have taken in recent decades. Hence, in the same way in which a single observation of the throwing of a dice only allows for a very limited understanding of the range of possible outcomes, a record of a climate observable often only allows for limited insights into the relative impact of external forcing or internal variability on the time evolution of that observable. In particular, all reliable observational records of sea ice only capture the behaviour of Arctic sea ice during a period of a rapidly changing background climate. Because of these rapid changes, even a 30-year long record is not long enough to provide a meaningful “average climate condition” that would sufficiently minimize the impact of internal variability , 5. TheiThe second limitation derives from observational uncertainty. In particular, all satellite records are based on indirect methods to obtain the underlying sea-ice property that one is ultimately interested in. Therefore, all major observational products are only an approximation of the true state of the sea-ice cover at any given time , 7.The third limitation derives from the fact that we can only observe a very limited subset of climate variables that matter for the evolution of sea ice. For example, we have only limited observations of the major atmospheric heat fluxes that determine the surface energy balance of the ice cover, and no Arctic-wide observations of the oceanic heat flux underneath the ice.2 emissions and sea-ice coverage identified in models and observations, where the slope of the linear relationship is taken directly from the observational record.If one keeps these limitations in mind, observational records can give us information about the sea-ice cover that offer a wealth of valuable insights into the past and also into the future evolution of Arctic sea ice. Indeed, we would argue that the most robust quantitative estimates of the future evolution of the ice cover are based on linear relationships between temperature or cumulative COThe studies that we summarize here are primarily based on the 40-year long record of gridded Arctic sea-ice concentration as obtained from passive-microwave satellite observations. This record started in the late 1970s, and is based on a series of successive multi-frequency passive microwave sensors that allow scientists to observe the polar regions year-round regardless of polar night or cloud cover , 9. The The studies that we discuss here primarily use the sea-ice area or the sea-ice extent derived from such satellite-retrievals of gridded sea-ice concentration. Arctic sea-ice area is usually calculated by multiplying sea-ice concentration with grid-cell area and adding up over all Northern-hemispheric grid cells. Arctic sea-ice extent is usually calculated by adding up the grid-cell area of all grid cells with at least 15% sea-ice coverage. In the following, we will use the term “sea-ice coverage” for any statement that is true for both sea-ice area and for sea-ice extent.Any climate model has limitations that must be kept in mind when employing it to understand the evolution of the sea-ice cover in the real world.2 emissions [First, climate models cannot capture all processes that govern the evolution of our climate system, and hence usually represent the real evolution of an observable less realistically than a given observational record. This is particularly true for sea ice, where climate-model simulations have been found to have substantial biases compared to the observed evolution of the ice cover. Among others, the models usually have a too low sensitivity of the simulated ice loss to simulated warming , 11–13 amissions , they ofmissions , they ofmissions , 17, andmissions , 19. TheSecond, the relationship of a model to the real world is often very difficult to estimate , which mOn the plus side, however, models overcome many of the mentioned limitations of observational records: models can be run several times and over long periods to capture the range of possible internal variability, they provide full three-dimensional fields of all major climate variables at nearly any desired temporal resolution, and they are internally consistent. In addition, they reflect possible future changes in the physical processes that drive the evolution of the sea-ice cover. Such future changes can obviously not be observed, which is why model simulations contribute important insights into the future evolution of the ice cover that can not be inferred from the observational record.Most recent studies that employ climate-model simulations to explore the ongoing and future evolution of Arctic sea ice are based on the coordinated set of simulations from about 40 different climate models that participated in CMIP5. In addition, some recent studies are based on large ensembles of simulations with individual models. Such single-model large ensembles have been used for sea-ice-related studies for the Community Earth System Model (CESM ), the CaChanges in the external forcing of the sea-ice cover have been established as the major driver of the observed loss of Arctic sea ice in the vast majority of related recent studies , 26, 27.2 concentration [2 emissions [2 are also the main driver of the observed warming of the atmosphere [2 emissions has been largely linear in the past.The major role of changes in the external forcing for the past evolution of Arctic sea ice has been identified both in the observational record and in model simulations. In particular, both in models and in observations the Arctic sea-ice coverage  during summer is linearly related to the rise in global-mean temperature , 30–32. ntration , 33 and missions , 34, 35.mosphere , and the2 emissions [2, R2 values range between 0.75 and 0.92 for every month of the year over the period 1953–2017 [2.The linear relationships between the external drivers and Arctic sea-ice coverage do not only hold during summer, but have been shown to hold for all months, both for temperature  and for missions . For the953–2017 . This suOther changes in external forcing have only had a limited impact on the sea-ice cover in the past few decades. For example, the observational record shows no substantial co-variation of Arctic sea ice with changes in solar activity . In partAlso anthropogenic aerosols have probably been of limited importance for the observed evolution of the Arctic sea-ice cover in recent decades. This is because their abundance has remained largely unchanged in the recent past, which is why it is unlikely that aerosols contributed much to the observed rapid loss of sea-ice evolution .The sensitivity of Arctic sea ice as described by the linear relationship between global-mean temperature and Arctic sea-ice coverage has been found to remain constant in model simulations across a wide spectrum of temperature trajectories , 32, 39.Unfortunately, as shown by the values for the September sea-ice cover listed in Table 2. Based on an average sea-ice extent during September of 4.7 million km2 and an average sea-ice area during September of 3.3 million km2 as given by the NSIDC sea-ice index for the past 10 years [2 is found to be between + 0.6 and + 0.9 ∘C relative to the average global-mean temperature of the past ten years  directly gives an uncertainty range of ± 330 Gt of cumulative CO2 emissions for the estimate of the occurrence of an ice free Arctic Ocean during summer. Hence, the 95% uncertainty range for cumulative future emissions of CO2 leading to an ice free Arctic Ocean during summer becomes 500 to 1100 Gt based on the average value of 800 Gt established above. For a given emission per year, this uncertainty range can be translated to an uncertainty range of the year when the Arctic first becomes ice free. For today’s emission of about 40 Gt CO2 per year, we get an uncertainty range of about 15 years, similar to the range of 20 years estimated in earlier studies based on model simulations [The observed relationship between Arctic sea-ice loss and cumulative COulations .In addition to changes in the external forcing and internal variability, also a possible self-amplification of the ice loss has been suggested to contribute to the substantial ice loss in recent years. This has often been framed in the context of nonlinear threshold behavior of the ice cover, also referred to as “tipping points.” The main mechanism that has been suggested to possibly give rise to such “tipping” of the ice cover has been the ice-albedo feedback.The observed evolution of the Arctic sea-ice cover is inconsistent with the possible existence of a tipping point arising from self-amplification of the ice cover. First, the evolution has remained linearly linked to the long-term rise in global-mean temperature as outlined above. Second, the 1-year lag autocorrelation of year-to-year changes in summer sea-ice coverage is negative , implyiSeveral stabilizing feedbacks during winter contribute to the lack of self-amplification even in light of the ice-albedo feedback : First, Note that all these stabilizing feedbacks are only active during winter. Throughout summer of a given year, any small perturbation of the ice cover will be effectively amplified. For example, the observed progressively earlier melt onset translates directly to more absorption of solar heat, and thus to a more substantial ice loss during summer and a later onset of autumn freeze-up. This behavior has been found in the observational record  and can The observed lack of self-amplification of the ongoing ice loss is consistent with the behaviour of the Arctic sea-ice cover in large-scale climate models. In contrast, simplified models of the sea-ice cover often show instabilities . This beAlso for the future, no substantial self amplification of the summer ice loss is expected. In particular, in all models that participated in CMIP5, the linear relationship between Arctic sea-ice cover and global-mean temperature holds until all sea ice is lost. This behavior already takes into account that in many regions, the ice-free duration during summer is becoming longer and longer, and that the ice cover as a whole is getting thinner. These factors are apparently not sufficient to overcome the stabilizing feedbacks and do not cause an acceleration of the summer sea-ice loss.In contrast, the pace of winter sea-ice loss accelerates once the total area covered in summer approaches nearly ice-free conditions, both in idealized studies  and in mAs summarized in the past sections, over the last few years, we have gained substantial understanding of the past and future evolution of the ice cover. The most robust of these insights have been derived from a combination of model simulations and observational records, primarily records of sea-ice concentration. Based on this recent progress, we believe that we have a good first-order understanding of the trajectory of the future Arctic Ocean towards a possibly seasonal ice cover. However, many of the details of this trajectory remain poorly understood. We see the following four issues as the most important open questions regarding our understanding of the past and future trajectory of the Arctic sea-ice cover.First, we lack understanding of the different pathways by which the ocean and the atmosphere translate changes in the external forcing and their own internal variability into changes of the ice cover. For example, the pathways by which heat is transported to the central Arctic Ocean are not clear. A recent study found that in 11 CMIP5 models, the heat required to melt the ice cover enters the Arctic via the atmosphere. In 11 other CMIP5 models, it enters the Arctic via the ocean, and in four CMIP5 models, both oceanic and atmospheric pathways supply net heat to the Arctic . This unSecond, we lack understanding of the regional evolution of the ice cover. For example, a regional analysis of trends in Arctic sea-ice coverage over recent decades has established substantial differences in response to the large-scale global warming , 70. To Third, we lack understanding of many of the implications of the ongoing ice loss. For example, a number of studies have suggested that the large-scale sea-ice loss might affect atmospheric circulation patterns and mid-latitude weather systems . In Fourth, we lack a good understanding of the past and future evolution of sea-ice volume. This is related to the lack of a sufficiently long and sufficiently reliable observational record of sea-ice volume, to uncertainties in reanalyzed sea-ice volume , and to In this review, we have demonstrated the consistency of existing observation-based studies with existing modeling-based studies regarding the future evolution of sea ice. In particular, we have demonstrated how we can use observational records to reproduce many recent quantitative estimates from primarily model-based studies. The quantitative link between models and observations is established as the agreement with the observed sensitivity has become a litmus test for any model-based estimate of the future evolution of the Arctic sea-ice cover. Where the observed sensitivity is not reproduced by a model, bias correction is applied to obtain reliable model-based estimates of the future evolution of the sea-ice cover. This also implies that often, the best estimate of the future evolution of the Arctic sea-ice cover is obtained by establishing robust relationships in models and observations, and to then extrapolate the quantitative manifestation of such robust relationships from the observational record into the future. The major uncertainty then derives from the quantification of the true sensitivity of the ice cover to changes in the external forcing.The Arctic sea-ice cover has been and will remain linearly related to global-mean air temperature in all months. Global-mean air temperature can hence be interpreted as the most important control variable on future Arctic sea-ice evolution.2 in more than 50% of all years for a global warming of less than 2 ∘C compared to pre-industrial levels.The observed linear relationship between Arctic sea-ice coverage and global-mean air temperature suggests Arctic sea-ice coverage to drop below 1 million km2 suggests Arctic sea-ice coverage to below 1 million km2 for more than 50% of all years for total future anthropogenic CO2 emissions of less than 800 Gt.The observed linear relationship between Arctic sea-ice coverage and cumulative anthropogenic emissions of CO2 for a given global-mean air temperature.From internal variability, September sea-ice coverage can vary by a maximum of ± 1 million km∘C for the global warming at which the Arctic Ocean loses its summer sea ice for the first time. For CO2 emissions, the uncertainty is about ± 300 Gt CO2 emissions. Hence, the Arctic Ocean can be expected to be nearly ice-free in 5% of all years for 500-Gt future CO2 emissions and in 95% of all years for 1100-Gt future CO2 emissions.This year-to-year fluctuation can directly be translated into an uncertainty of ± 0.2 to 0.4 2 emissions.As the observed linear relationship between Arctic sea-ice coverage and global-mean temperature currently hold for all months, they allow us to estimate the future seasonal cycle directly from the observational record. This is also true for the observed linear relationship between Arctic sea-ice coverage and cumulative COBased on this approach, we identify the following statements to be very likely true for the future evolution of the Arctic sea-ice cover: 2 per year, these findings imply a substantial likelihood of an ice-free Arctic Ocean during summer before mid-century. The time window to prevent the loss of the Arctic summer sea-ice cover hence closes very rapidly.Based on current emission rates of about 40-Gt CO


It is now well established that cAMP can mediate a plethora of functional effects via a complex system of local regulatory mechanisms that result in compartmentalized signalling. The use of fluorescent probes to monitor cAMP in intact, living cells have been instrumental in furthering our appreciation of this ancestral and ubiquitous pathway and unexpected details of the nano-architecture of the cAMP signalling network are starting to emerge. Recent evidence shows that sympathetic control of cardiac contraction and relaxation is achieved via generation of multiple, distinct pools of cAMP that lead to differential phosphorylation of target proteins localized only tens of nanometres apart. The specific local control at these nanodomains is enabled by a distinct signalosome where effectors, targets, and regulators of the cAMP signal are clustered. In this review, we focus on recent advances using targeted fluorescent reporters for cAMP and how they have contributed to our current understanding of nanodomain cAMP signalling in the heart. We briefly discuss how this information can be exploited to design novel therapies and we highlight some of the questions that remain unanswered. The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is involved in multiple physiological functions. In response to a variety of different hormones and other extracellular cues that operate via activation of G protein-coupled receptors (GPCRs), cAMP affects cellular processes that range from modulation of metabolic enzymes to regulation of transcription, from the control of cell survival to cross-talk with multiple other signalling pathways. In eukaryotic cells, cAMP activates three ubiquitously expressed intracellular cAMP effector proteins, the cAMP-dependent protein kinase A (PKA), the cyclic nucleotide gated ion channels, and the exchange protein activated by cAMP (Epac) . The pref channels by cAMP and PKA-dependent phosphorylation of L-type Ca2+ channels (LTCC) at the sarcolemma (SL) and phosphorylation of ryanodine receptors (RyRs) and phospholamban (PLB) at the sarcoplasmic reticulum (SR) results in increased amplitude of the Ca2+ transient and enhanced heart rate (chronotropy) and strength of contraction (inotropy); by promoting Ca2+ reuptake into the SR, PKA-dependent phosphorylation of PLB also enhances relaxation (lusitropy); at the myofilaments (MF), PKA phosphorylation of troponin I (TPNI) reduces MF Ca2+ sensitivity and phosphorylation of myosin-binding protein C (MyBPC) increases the speed of cross-bridge formation, resulting in enhanced cardiac relaxation and contraction, respectively [In the heart, cAMP signalling mediates the sympathetic fight-or-flight response by regulating heart rate and strength of contraction via modulation of several aspects of the excitation–contraction coupling (ECC) process. ECC involves activation of β-adrenergic receptors (β-ARs) by catecholamines followed by synthesis of cAMP, activation of PKA and phosphorylation of a numbers of key protein targets. Direct activation of Iectively . As thisUnderstanding how cardiac myocytes respond specifically to different stimuli that signal via cAMP and PKA has been the focus of intense investigations in recent years. The current view is that regulation of this intricate signalling system relies on compartmentalization of cAMP at the nanoscale level, a process that results in the tailored local concentration of the second messenger to achieve selective activation of downstream targets. The development of fluorescent biosensors for real-time detection of cAMP in intact cells has been instrumental in unravelling the compartmentalized nature of cAMP signalling and recently developed targeted reporters are providing key information on the organization and regulation of cAMP subcellular nanodomains . A cAMP Research in the last two decades has clearly established that compartmentalization plays a key role in determining the specificity of cAMP/PKA signalling. This was first hypothesized on the basis of the observation that despite generating similar elevation of global cAMP levels, catecholamines mainly activate type II isoforms of PKA and promote cardiac inotropy and lusitropy, whereas prostaglandins activate type I PKA and have no effect on cardiac muscles contraction . To explThere is now ample evidence to support this model. Studies have shown that compartmentalized cAMP/PKA signalling is achieved via spatial segregation of the molecular components that carry out these signalling events into distinct signalosomes . Typical+ current [2+ influx through LTCC [2+ reuptake via selective phosphorylation of PLB [In ventricular myocytes, several AKAP complexes have been identified in different subcellular domains. For example, Yotiao, also known as AKAP9, forms a complex with AC2/9, the potassium channel subunit KCNQ1 and the phosphatase PP1 at the SL and this complex is necessary for cAMP-dependent regulation of K current ; the AKAugh LTCC –14; the n of PLB ,16; an mn of PLB ; at the n of PLB , α-synemn of PLB , and myon of PLB  have been of PLB ,22.Development of fluorescence resonance energy transfer (FRET)-based reporters has made it possible to directly image compartmentalized cAMP in living cells through direct visualization of local changes in cAMP levels. The possibility to monitor cAMP signalling as it happens in intact, living cells, where the complexity of the intracellular architecture and the spatial organization of the biochemical machinery is preserved, has been instrumental in defining the compartmentalized nature of cAMP signalling and is starting to provide unexpected, novel insight into cell physiology. Targeting these sensors to specific subcellular locations is proving particularly fruitful.A successful family of FRET-based reporter used the cAMP effector protein Epac or its cyclic nucleotide-binding domain (CNBD) sandwiched between yellow fluorescent protein and cyan fluorescent protein (YFP–CFP) –25. The cAMP universal tag for imaging experiments), and engineered to minimize such interference. CUTie includes the CNBD B from the regulatory subunit type IIβ of PKA as the cAMP sensing moiety. Similarly to the earlier targeted Epac-based sensors, CFP is fused to the carboxyl terminus of the CNBD but the YFP moiety is removed from its amino-terminal end. Sequence conservation analyses and molecular simulations determined that the insertion of the YFP in an intra-domain loop of the CNBD (loop 4–5) was compatible with the correct folding of both the CNBD and the YFP. The advantage of this molecular architecture is that it leaves the amino-terminus of the CNBD free for fusion to arbitrary targeting domains, while the allosteric reactions remain confined to a hinging point near the lid of the cAMP binding pocket  or lower (at TPNI on MFs) than the concentration of the second messenger in the bulk cytosol. These findings shift the focus from a model of restricted propagation of cAMP from the site of synthesis to the bulk cytosol to one where the level of cAMP is locally regulated within individual nanodomains. According to this paradigm, cAMP can freely diffuse from the plasma membrane to the entire cell but the functional outcome of the cAMP signal is dictated by the local levels of the second messenger achieved in the extremely restricted environment immediately surrounding specific effector/target protein complexes . At somein situ re-association of C subunits. This would limit the distance from point of release over which C subunits can phosphorylate substrates, a mechanism that would further support compartmentalized phosphorylation of local targets [Although experimental evidence shows that cAMP levels at some subcellular sites can increase above the level in the bulk cytosol ,9 the me targets . Alterna targets . The abo targets .Figure 2+ transient and contraction, whereas PKA-mediated phosphorylation of TPNI reduces myofilament Ca2+ sensitivity and limits contraction. Therefore, a delayed and attenuated cAMP signal at the troponin complex may be necessary to fine tune the effect of catecholamines and avoid that the opposing effects of PKA on calcium handling counteract each other. Interestingly, PDE inhibitors used in the clinic for the treatment of heart failure lead to increased mortality in the long term. By obliterating the difference between cAMP nanodomains and by reducing the efficiency of contraction, non-selective PDE inhibition may curtail the benefit of local control of cAMP and a larger cAMP signal at TPNI may effectively deplete diastolic cardiac reserve via Ca2+ desensitization.An unexpected finding using targeted versions of the CUTie reporter was that subcellular sites that contain PKA targets known to be activated by catecholamines in the fight-or-flight response, and therefore expected to belong to the same functional domain, in fact, experience cAMP signals of different amplitude and kinetics. As mentioned above, the data show that the cAMP signal detected in the environment surrounding the troponin complex at the MF is significantly smaller and delayed compared to the signal detected at the AKAP18δ/SERCA/PLB complex at the SR or the AKAP79/β-AR/AC/LTCC complex at the SL. Active PDEs are required for the signal to be compartmentalized, as the targeted sensors detect identical signals at the three sites when the PDEs are inhibited in the presence of isobutyl-metylxanthine, a non-selective PDEs inhibitor. Inhibition of PDEs, which results in a homogeneous increase in cAMP, also results in reduced myocyte shortening indicating that spatial coordination of distinct cAMP signals is required to achieve maximal efficiency of regulated contraction and relaxation. Why is the compartmentalized cAMP signal more effective than a uniform cAMP signal? The effect of PKA-dependent phosphorylation of LTCC and PLB is to enhance the Ca2+ sensitization and diastolic dysfunction. In other words, the evidence suggests that the tighter regulation of cAMP levels at the troponin complex results in optimal enhancement of contraction and relaxation but this comes at the cost of intrinsic vulnerability of the myofilaments to Ca2+ sensitization, which may become apparent in conditions of reduced adrenergic drive.Although required to maximally enhance contraction and relaxation, attenuation of cAMP signals at the troponin complex comes with a downside. Experiments using animal models of cardiac hypertrophy and heart failure, which are characterized by down-regulation of the adrenergic system and reduced synthesis of cAMP, show that in hypertrophic/failing cardiac myocytes the cAMP signal at TPNI is dramatically reduced compared with the other sites, to a level that may not result in PKA activation. This may result in myofilament Ca2+ release from MFs in the presence of β-blockers would result in exacerbation of the functional deficit.These findings may also provide a rationale for the limited success of β-blocker therapy in heart failure with preserved ejection fraction (HFpEF), a condition that comprises almost half of the population burden of heart failure and for which there is currently no effective treatment . HFpEF i2+ sensitization in pathological conditions. The possibility to treat disease with precision by targeting individual cAMP domains is exciting but an exhaustive molecular understanding of cAMP nanodomains localization, regulation and function is required before this potential can be exploited.The novel insight provided by targeted reporters into the details of local cAMP signalling has important implications for the development of new therapeutic strategies ,48. For Importance of the field: It is now well established that cAMP mediates a plethora of functional effects while maintaining the fidelity of response via compartmentalization. FRET-based probes for cAMP, which can detect compartmentalized cAMP in intact cells with unprecedented accuracy, have been instrumental in defining the role of compartmentalized signalling. Recent developments using targeted probes to study cAMP signalling in cardiac myocytes are starting to provide novel mechanistic insight into cell physiology and its alterations in pathological conditions. These findings have important implication for the development of novel therapeutic strategies.Summary of current thinking: Recent evidence suggests that a delayed and attenuated cAMP signal at the troponin complex may be necessary to fine tune cardiac contraction and relaxation in response to catecholamines. Active PDEs are required for this compartmentalized signal since inhibition of PDEs results in a homogeneous increase in cAMP throughout the myocyte and reduced myocyte shortening. The current view is that the specific local control at these nanodomains is enabled by a distinct signalosome where effectors, targets, and regulators of the cAMP signal are clustered. Data show that the size of these domains can be as small as a few tens of nanometres, indicating that the functionally relevant cAMP signal is provided by the local concentration of cAMP in the space immediately surrounding the effector PKA within the signalosome, rather than by the global change in cAMP. At some sites, local PDEs act as sinks for cAMP, keeping the level of the second messenger below the bulk cytosol level. At other sites, cAMP raises above the bulk cytosol level through mechanisms that may involve low local PDE activity and high concentration of cAMP binding domains. This arrangement may prevent the dissipation of the local signal by ‘trapping’ cAMP within the nanodomain.Future directions: The current challenge in the field is to determine a comprehensive map of subcellular cAMP nanodomains and to understand the details of their molecular composition, regulation, and function. The recent findings in cardiac myocytes illustrate how an understanding of these molecular details may provide an opportunity to develop precision medicine approaches. The unique cAMP signalling occurring at the myofilament in response to sympathetic stimulation relies on local specific PDE isoforms. The identification of these enzymes will allow their selective targeting and may provide an effective means to re-constitute the appropriate level of cAMP signal in cardiac pathological conditions where calcium sensitization is affecting function. Further studies with targeted reporters will undoubtedly contribute to advance our understanding of the molecular basis of functional cAMP nanodomains, in the heart as well as in other organ systems.


Necessitated by the subzero temperatures and seasonal exposure to ice, various organisms have developed a remarkably effective means to survive the harsh climate of their natural habitats. Their ice-binding (glyco)proteins keep the nucleation and growth of ice crystals in check by recognizing and binding to specific ice crystal faces, which arrests further ice growth and inhibits ice recrystallization (IRI). Inspired by the success of this adaptive strategy, various approaches have been proposed over the past decades to engineer materials that harness these cryoprotective features. In this review we discuss the prospects and challenges associated with these advances focusing in particular on peptidic antifreeze materials both identical and akin to natural ice-binding proteins (IBPs). We address the latest advances in their design, synthesis, characterization and application in preservation of biologics and foods. Particular attention is devoted to insights in structure-activity relations culminating in the synthesis of de novo peptide analogues. These are sequences that resemble but are not identical to naturally occurring IBPs. We also draw attention to impactful developments in solid-phase peptide synthesis and ‘greener’ synthesis routes, which may aid to overcome one of the major bottlenecks in the translation of this technology: unavailability of large quantities of low-cost antifreeze materials with excellent IRI activity at (sub)micromolar concentrations. Ice-binding proteins (IBPs) comprise a unique class of biomacromolecular ice crystal growth modifiers that offer their host a means to thrive in extreme environmental niches. As unique binders of ice crystal planes, IBPs aid survival in sub-zero ice-laden habitats in various ways ranging from ice adhesion to stay put in a nutrient-rich environment  to inhibIBPs expressed in microbes, plants, fish, algae and insects are both structurally and functionally diverse. To date, five biological functions have been characterized: freezing point depression , inhibitwfAFP  are aligned within the helical axis and the oxygen-oxygen spacing between hydroxyl groups is 16.1 Å, 16.0 Å and 16.2 Å, respectively [Computational studies showed that the hydroxyl groups of the four Thr residues in the ectively . This maTenebrio molitor has several isoforms of the protein TmAFP [Fish AFPs do not contain β-helices, but the structural motif is abundant in AFPs from insects, bacteria and over-wintering plants. The beetle in TmAFP , all of sbwAFP contains repeating patterns of 15 amino acids organized in a β-helix structure with a triangular cross-section and arrays of TXT motifs at the flat IBS [Rhagium inquisitor. The RiAFP , wherein X is a residue with a polar side chains [The IBS consists of regular arrays of TXT motifs positioned on one side of the helix. Similarly, the spruce budworm flat IBS . The dishe RiAFP b IBS con β-helix . The ice perenne c folds ae chains ,28, therO-glycosylated at the threonine side chains with the disaccharide β-D-galactosyl(1–3)-α-N-acetyl-D-galactosamine  helices are displayed in insect AFPs without α- nor β-helices, such as the snow flea AFP a. Molecuvia their IBS. Growth ceases at the location of the bound AFPs, but continues in between the adsorbed proteins until the addition of water molecules to the convex surface is no longer energetically favorable. Ice growth is now completely halted and the freezing point depressed. Growth remains prohibited as long as the temperature remains above the non-equilibrium freezing point, which depends on the magnitude of the local curvature set by the spacing between bound proteins and the undercooling Ps to function. Yet, the physico-chemical principles of the interaction between AF(G)Ps and ice remain elusive, as both experimental and theoretical studies of protein binding to ice are challenging. In recent years, computational studies ,35,36,37rcooling . In the DAFP-1 and the MpAFP from the Antarctic bacterium Marinomonas primoryensis bind up to three different crystallographic planes. These proteins bind with high affinity to the basal plane (0001) and can also recognize both the primary and secondary prism (1120) [Ice-binding proteins binds ice, but not in the same manner. Preferential adsorption of IBPs onto specific sets of ice crystal planes a in specm (1120) ,50. Tf, Tm) in which nucleated ice crystals cease to grow. These blocks further growth of internalized ice in e.g., the intestinal tract of fish living in ice-laden waters. The magnitude of the generated TH gap  is strongly dependent on AF(G)P type and concentration [Fragilariopsis cylindrus, which display moderate freezing point depression yet suppress basal plane ice growth [Critical for the survival of freeze-avoidant fish and insects is the thermal hysteresis (TH) or antifreeze activity of antifreeze (glyco)proteins. Ice-binding of AF(G)Ps generates a so-called TH gap between a non-equilibrium freezing and melting point  made a wide variety of peptidic antifreezes, such as cyclic peptides and glycopeptides ,79,80, swfAFP and AFGPs like AFGP8 from polar fishes, which are amenable to both solution phase synthesis and solid-phase peptide synthesis (SPPS). Chemical ligation has been employed to produce larger insect AFPs based on the beetle Dendroides canadensis from smaller fragments produced by SPPS.To date, chemical synthesis strategies have focused primarily on the smallest IBPs with a highly repetitive amino acid sequence and a relatively simple secondary structure. These are type I AFPs like wfAFP mutants have been prepared by SPPS, wherein one or more threonine residues in the IBS carrying both OH and CH3 functionalities have been replaced by amino acids with either pendant OH groups (serine) or CH3 groups  [wfAFP. The methyl functionalities may bind inside the holes in the middle of six-membered water rings in the ice lattice, as observed for methyl groups of specific hydrophobic amino acids including Thr-18 in the IBS of ocean pout type III AFP [TmAFP reveal a distinct role for both hydrophobic and hydrogen-bonding groups in its IBS. Amongst other relevant actions, the methyl groups stabilize the anchored clathrate-like waters at the IBS and the hydroxyl functionalities dock it onto ice [wfAFP led to the complete loss of thermal hysteresis activity in an A17L-wfAFP mutant [To shed light on the relative importance of hydrophobicity and hydrogen bonding for ice-binding, various  ,81. The  . Substit III AFP . Moleculonto ice . AnotherP mutant .Antifreeze glycoproteins are notoriously difficult to synthesize and characterize, but the challenge has been addressed successfully by several groups . NishimuDendroides canadensis AFP [DcAFP were synthesized by SPPS. Next, the formation of disulphide bridges was promoted to ensure correct folding of the protein, which was confirmed by 1H-NMR. After Cys-Cys bridge formation, the different fragments were connected by chemical ligation in a step-wise fashion to synthesize the native protein. The same strategy was used to create a non-natural variant with lactam bridges instead of disulphide bridges upon substitution of the cysteine residues with lysine and aspartic acids. Importantly, this approach did not only improve the synthetic yield and purity, but the novel AFPs were also more stable than their natural disulphide bridge containing counterparts. The thermal hysteresis activity of the two constructs was similar, with TH values at a protein concentration of 10 mM between 0.1 and 0.35 °C [The largest chemically synthesized AFPs were based on the structure of the beetle nsis AFP . First,  0.35 °C .De novo antifreeze analogues offer unprecedented means to advance our understanding of the working mechanism of ice-binding proteins and may be prepared in a more straightforward and less time-consuming manner than native AFPs. Different orthogonal chemical ligations not present in natural AF(G)Ps have been investigated to improve synthetic accessibility and probe whether it impacts antifreeze activity. Constructs varying considerably in composition and architecture from native AFGPs have been investigated to identify the minimal requirements for activity and the impact of various structural elements, such as secondary folds, tethered sugars, and free N- and C-termini.wfAFP, these constructs had a high content of Ala facilitating the formation of an α-helical fold. Unlike wfAFP, these constructs were devoid of the threonine residues supposedly essential for ice binding. Instead, the constructs contained lysine residues to improve solubility in water. Surprisingly, despite the absence of Thr, the peptidic constructs exhibited thermal hysteresis activity, albeit low with a modest TH of 0.3 °C at 50 mM in 0.1 M ammonium bicarbonate buffer [sfAFP, the right-handed amino acid sequence by employing chemical ligations. The sequential ligation was done with N-terminal cysteines incorporated as 1,3-thiazolidine-4-R-carboxylic acid (Thz) and a thioester at the C-terminal [Inspired by type I AFPs, Wierzbicki and coworkers prepared a series of de novo constructs by SPPS comprising 43 amino acid long polypeptides with repetitive sequences of alanine and lysine residues . Like wfe buffer . Glycineterminal . Aiming Proline is unique amongst the canonical amino acids in that it has no amide N–H. Consequentially, polyproline cannot form intramolecular hydrogen bonds. In combination with the pyrrole functionality, this makes the polypeptide water-soluble and quite hydrophobic at the same time. Graham and coworkers hypothesized that polyproline helices can be considered the minimal AF(G)P mimic due to their implicit amphiphilicity. To demonstrate that, they performed a series of experiments with polyproline constructs of different length and stereochemistry . These pDrori and coworkers recently investigated the activity of self-assembling organic dyes that display specific functional groups in a periodic manner resembling the periodic display of functional groups in IBPs like AFGPs. The organic dye Safranine O was able to show TH activity of −0.32 °C at a concentration of 28 mM, a value comparable to AFGP8  . MoreoveN-acetyl-D-galactosamine attached to the threonine were coupled using liquid phase peptide synthesis. To cyclize the linear peptides, the solution was diluted 100 times and fresh coupling reagent was added to the mixture. Interestingly, these cyclic analogs showed ice shaping properties and TH activity similar to their linear counterparts [To test if the amino and carboxy group of AFGPs play a role in ice binding, Hachisu and collaborators prepared hexa-, nona- and dodecacyclic AFGP analogues. 2, 3 or 4 ATA repeats with the disaccharide β-D-galactosyl(1-3)-α-terparts . The TH terparts . TmAFP, which presents a β-helical rich structure where threonine amino acids are organized in a regular array of TXT. Previous experiments and simulations showed how cyclic peptides of alternating L- and D- amino acids self-assemble into nanotubes under various conditions [α-Cα intramolecular distance of 7.04 Å, and a Cα-Cα intermolecular of 4.88 Å. These values are close to the values shown for TmAFP  and the distances of hexagonal ice (7.356 Å and 4.518 Å). Molecular dynamics (MD) simulations also revealed a flat surface in the Thr-ala-Thr region, where on average more than one water interacts with the hydroxyl groups of the threonine residues through H-bonding.Their design was based in the insect antifreeze protein nditions ,100,101.de novo antifreezes [Virtual screening techniques, such as the basic local alignment search tool (BLAST) and quantitative structure-activity relationship (QSAR) models, have become an important tool in drug discovery ,103 aidiifreezes ,102. Yang and coworkers compiled an effective predictor of new AF(G)Ps based on protein similarity using BLAST. A training dataset and an independent dataset consisting of 481 AFPs and 9,193 non-AFPs were used to create the basis of the predictor. These datasets contained all the diverse primary sequences and structures of known AF(G)Ps. The predictor used features of the AFP such as functional domain, the evolutionary conservation of these domains across proteins of the same family and statistical cross-validation to classify the best protein candidates that may show TH .Ben′s group used a set of 124 previously synthesized compounds with known IRI activities to calibrate a 3D-QSAR model . Input tcAFP. In addition, 5 proteins that show no TH but have exposed planar surfaces and/or secondary structures similar to AFPs were used as a negative control set. The generated model successfully predicted the antifreeze activity of known AFPs not included in the training set based on the assumption of a linear relation between TH activity and √cAFP [Kozuch and collaborators combined molecular dynamics of water molecules near an AFP surface and geometric structure of the IBS with neural networks aiming to predict the magnitude of the thermal hysteresis gap of AFPs . To builnd √cAFP . The devApplied interest in IBPs sparked soon after the discovery of the first AFGPs approximately 50 years ago and has continued to blossom in recent years into a highly active area of IBP research. This is because the protective properties displayed by IBPs are not only interesting for biomedical fields such as cryopreservation and cryosurgery ,77,107, Biomedical application of AF(G)Ps and their mimics is amongst the most active areas of IBP research. The interested reader is referred to recent reviews on the matter addressing in particular polymeric mimics , peptidiLactococcus lactis for the production of large amounts of type I AFP. The lyophilized crude AFP was used to treat meat and dough to improve the post freeze-thaw structure, taste and composition. Frozen meat treated with type I AFP suffered less from protein loss and scored higher values in a sensory evaluation of juiciness. Dough treated with a crude lyophilized powder of type I AFPs showed better fermentation capacity than non-treated frozen dough [DcAFP altered the structure, cooking properties, and texture of freeze-thawed carrots [DcAFP analogues for 7 days reduced drip-loss 5-fold. Freezing degraded the texture of both treated and untreated samples relative to the fresh vegetable, but not to the same extent. AFP-treated samples scored considerably better in the two key texture parameters for consumer preference: hardness and firmness. A comparative analysis by cryo-SEM revealed the microstructural changes underlying these positive effects. Due to the growth of larger (intracellular) ice crystals in the untreated samples, these display larger and more intercellular spaces, as well considerable cell membrane and cell wall deterioration. Starch, emulsifiers, proteins, and various other hydrocolloids are routinely used to maintain and stabilize the structure of food networks and emulsions. Likewise, AFPs and their analogues have been added to frozen foods to mitigate freeze-thaw induced alterations in texture and associated sensory properties. Peng and co-workers developed a food-grade expression and fermentation system based in protein expression in en dough . Hydrolyen dough . The losen dough . Treated carrots . Drip loMaterials and tools have become available to elucidate the structure-function relations of the broad spectrum of structurally and functionally diverse ice-binding proteins that have been discovered. Long-standing open questions related to the working mechanism of ice-binding proteins may now be addressed both computationally and experimentally. Computationally guided design approaches and further optimization of synthetic strategies will facilitate further development of novel IBP analogues. Various ice-binding motifs may be incorporated into a single analogue to maximize efficacy. Assays of e.g. ice recrystallization activity could be further standardized to facilitate a fair comparison between the activiy of diverse classes of IBPs and their analogues. We also anticipate more in-depth studies on the application of IBPs and analogues in non-native environments geared towards an improved understanding of the factors that impact performance, so as to inform the selection of an appropriate IBP and/or the modular design of a suitable analogue. For example, we have recently investigated the impact of salts which are commonly present in cryopreservation media on the ice recrystallization activity of native AF(G)Ps. Interestingly, the study revealed that sodium borate has little impact on IRI-activity, while it dramatically reduced TH activity . These f2Cl2). Albericio´s team substituted DMF by THF or acetonitrile (MeCN) [Synthetic approaches to produce peptidic antifreezes by solid-phase peptide synthesis can be further optimized to maximize yield, purity, and time-efficiency without generating large amounts of toxic waste, which constitute an environmental burden and health risk as chronic diseases may arise upon continued exposure. This motivated various successful attempts to adapt current protocols to utilize more environmentally benign solvents and coupling agents. Aiming to find a greener and safer alternative for DMF, McMillan and collaborators studied the effect of various solvents on amide bond-forming reactions . Their re (MeCN) . Synthese (MeCN) ,120. ThiRoughly 50 years since the first experiments on freezing-point depression by (glyco)proteins, the field of ice-binding proteins continues to flourish. The continued involvement of researchers from various backgrounds has stimulated scientific discussions, generated new research directions, and advanced both fundamental insight and efforts targeting utilization of antifreezes in various applications. Herein we have reviewed recent progress in IBP research focusing on peptidic antifreezes. We addressed their design, synthesis, characterization and application in preservation of biologics and foods. The widespread availability of diverse classes of IBPs, state-of-the-art experimental tools and computational methods may culminate in the near future in the elucidation of structure-activity relations aided by new developments in analogue synthesis, computational studies by molecular dynamics and quantum chemical calculations, and single-molecule experimentation. Bioinspired molecular designs of novel compounds will offer new insights into molecular recognition, as well as the relation between TH and IRI activity. The structural diversity of native IBPs suggests that it is possible to design a platform of new antifreeze materials varying in (amino acid) composition and higher order structure. Multiple putative ice-binding sites may also be combined into a single mimic aiming to bind to various ice crystal planes and enhance activity. Activity geared towards applications, especially in biomedicine, will continue to grow. We anticipate that the implementation of increasingly systematic and multidisciplinary approaches will bear fruit and ultimately deliver on the promise of IBPs as cryoprotectants in non-native environments.


The cellular levels and activities of ribosomes directly regulate gene expression during numerous physiological processes. The mechanisms that globally repress translation are incompletely understood. Here, we use electron cryomicroscopy to analyze inactive ribosomes isolated from mammalian reticulocytes, the penultimate stage of red blood cell differentiation. We identify two types of ribosomes that are translationally repressed by protein interactions. The first comprises ribosomes sequestered with elongation factor 2 (eEF2) by SERPINE mRNA binding protein 1 (SERBP1) occupying the ribosomal mRNA entrance channel. The second type are translationally repressed by a novel ribosome-binding protein, interferon-related developmental regulator 2 (IFRD2), which spans the P and E sites and inserts a C-terminal helix into the mRNA exit channel to preclude translation. IFRD2 binds ribosomes with a tRNA occupying a noncanonical binding site, the ‘Z site’, on the ribosome. These structures provide functional insights into how ribosomal interactions may suppress translation to regulate gene expression. Translation is an important point of regulation for gene expression. The overall levels and activities of ribosomes are implicated in cellular differentiation, developmental disorders, and cancers . Red bloUnderscoring the importance of ribosomal activity in blood cell differentiation, mutations in ribosomal proteins, ribosome-assembly factors, and the ribosome-degradation machinery manifest as blood disorders  and are Although controlling absolute ribosome levels is an effective mechanism to regulate gene expression, assembling and degrading ribosomes is slow and requires significant energy consumption . For fasDespite emerging evidence of the importance of global translation levels on short- and long-term gene regulation, other mechanisms for modulating ribosome activities remain poorly characterized. Here, we use electron cryomicroscopy (cryo-EM) to analyze translationally inactive ribosomes isolated from mammalian reticulocytes, the penultimate stage of red blood cell differentiation . In addiWe previously used a cell-free translation system derived from rabbit reticulocyte lysate to isolate various ribosomal complexes for cryo-EM analysis . DespiteThree-dimensional classification of the combined dataset of ribosomal particles identified nine structurally distinct classes of 80S ribosomes ; Table 1The two most abundant classes of non-translating ribosomes are distinct conformations (rotated and unrotated) of 80S ribosomes bound to the elongation factor eEF2 . eEF2 no10-helix around nucleotide C1701 of 18S rRNA , which follows an unstructured N-terminus absent in our reconstruction, adopts an Armadillo-type fold formed by six HEAT repeats . Each HEThese interactions indicate that IFRD2 engages non-translating ribosomes with vacant mRNA channels and precludes further translation. Although a previous large-scale mass spectrometry study suggested that the ribosomal interaction of IFRD1 and IFRD2 are sensitive to RNase treatment , treatinThe observation that almost 8% of particles in the dataset contain IFRD2 suggests that it is highly abundant in reticulocytes. Indeed, IFRD2 appears to be ~8 fold less abundant on human HEK293T ribosomes compared to reticulocyte ribosomes . OverexpAll ribosomes bound to IFRD2 in the reticulocyte dataset also contain a tRNA in an extreme position on the ribosome past the E site that we call the ‘Z site’ . AlthougLike the universal tRNA-binding positions, Z-site tRNA bridges the ribosomal subunits. Three distinct interactions are observed between the tRNA and the large subunit. First, the backbone of the 3’ CCA binds a lysine-rich stretch of eL42 (residues 27 – 30) . Second,Our structural analysis of reticulocyte ribosomes in a large cryo-EM dataset has revealed new insights into ribosome inactivation in mammals. We identify new similarities between yeast Stm1 and mammalian SERBP1, reveal the interactions that sequester eEF2 on the ribosome, and redefine the IFRD family as ribosome-silencing proteins.As proposed for Stm1, IFRD2 may repress translation during cellular stress. Previous studies indicate that IFRD2 expression is induced in specific circumstances, such as tetradecanoyl phorbol acetate treatment and serum replenishment after starvation . In addiOur results suggest that IFRD2 is especially abundant on ribosomes in reticulocytes, the immediate precursors to red blood cells. Reticulocytes also express enhanced levels of other factors that can suppress protein synthesis, including the heme-dependent eIF2α kinase HRI . ConsideWhile SERBP1 and IFRD2 may act redundantly to inactivate mammalian ribosomes, our structures suggest they target different subsets of ribosomes and may induce different fates. In yeast, Stm1-inactivated ribosomes are recycled after starvation by Dom34-Hbs1  and Rli1  . WhetherWe additionally observe Z-site tRNA on all reticulocyte ribosomes containing IFRD2. Although we cannot rule out the possibility that a tRNA in the Z site results from an artefact of isolating these samples, the stable association of Z-site tRNA and the similarity with IRES interactions supports the idea that the Z site is a preferred binding site for certain RNA structures, potentially including cellular mRNAs. In yeast, knocking out eS25, a key interacting partner of Z-site tRNA, decreases global protein synthesis and slows growth . A recenThe Z-site may represent a late-stage intermediate of tRNA ejection downstream of the E site. This hypothesis is supported by the observation that Z-site tRNA can bind simultaneously with a P-site tRNA but not a canonical E-site tRNA, and that Z-site tRNA interacts with the L1 stalk, which interacts with all known tRNA-ejection intermediates in bacteria  Zhang e. HoweverA Z-site tRNA ejection intermediate would be transient during active translation, occurring only before the next translocation event. Therefore, the presence of a stably bound Z-site tRNA could act as signature of a translationally incompetent ribosome regardless of whether it engages the Z site from the E site or from a pool of deacylated tRNAs in the cytosol. In this scenario, tRNA binding to the Z site is more likely when deacylated tRNA levels rise, for example by amino acid depletion, or when translational factors are limiting. Intriguingly, deacylated tRNAs, a yeast-specific tRNA ejection factor, and stalled ribosomes are all implicated in regulating the integrated stress response by the GCN2 kinase .Our unexpected observation of IFRD2 and a new tRNA position on the mammalian ribosome highlights the ability of mining cryo-EM datasets to reveal new biological interactions from heterogeneous samples. This strategy is directly applicable to identifying and visualizing binding partners of other biological assemblies.The ORF of IFRD2 was subcloned into a pcDNA3.1-based vector after a CMV promoter and an N-terminal 3X Flag tag and TEV cleavage site. IFRD2 antibody was obtained from Invitrogen (PA5-48833). Anti-FLAG M2 monoclonal antibody (F3165), resin (A2220) and 3X FLAG peptide (F4799) were obtained from Sigma-Aldrich.2, 0.1% Triton X-100, 1 mM DTT, (2) 50 mM Hepes pH 7.4, 250 mM KOAc, 5 mM Mg(OAc)2, 0.5% Triton X-100, 1 mM DTT, and (3) 50 mM Hepes pH 7.4, 100 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT. Two sequential elutions were performed with 0.1 mg/mL 3X Flag peptide in the third wash buffer for 25 min each at room temperature. This elution was directly analyzed as ‘sample A’  and analyzed by SDS-PAGE and Coomassie staining. The entire gel lanes were excised and submitted for mass spectrometry analysis as ‘sample B’  and ‘sample C’ . The proteins identified by mass-spectrometry are given in To reduce the identification of ribosomal protein peptides, we evenly divided the elutions into two halves. We maintained one half in physiological salt concentrations and adjusted the other half to a final concentration of 750 mM KOAc, 15 mM Mg(OAc)2, 0.5% Triton X-100, 1 mM DTT, and protease inhibitor cocktail. Lysates were clarified by spinning at 20,000xg at 4°C for 10 min before being subjected to biochemical treatments.HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose and 10% fetal bovine serum and verified to be mycoplasma free. Transfections of Flag-tagged IFRD2 were performed when cells were 60 – 70% confluency using TransIT 293 (MIRUS) according to manufacturer instructions. 16 – 18 hr after transfection, we lysed cells in 50 mM Hepes pH 7.4, 100 mM KOAc, 5 mM Mg(OAc)2 without or with 10 mM EDTA.Where applicable, rabbit reticulocyte lysate  for 30 min at 4°C using the slowest acceleration and deceleration settings. Eleven 20 μL fractions were manually collected from the top for immunoblotting analysis.Four 10 cm plates of HEK293T cells were transfected as described above and passaged 1:4 the day after transfection. Two days later, the cells were lysed as described above and incubated with M2 anti-Flag agarose resin for 1 hr at 4°C. The resin was washed with 6 mL of lysis buffer and subjected to two sequential elutions using one column volume of 0.1 mg/mL 3X Flag peptide incubated at room temperature for 25 min. A portion of the elution was directly frozen onto cryo-EM grids, and the leftover elution was pelleted at 100,000 rpm for 40 min at 4°C in a TLA120.2 rotor to isolate ribosomes, which were subjected to biochemical analysis .For cryo-EM analysis, 3 μL of the pooled elution containing ~80 nM ribosomes were directly frozen onto glow-discharged Quantifoil Cu R2/2 grids with a thin (~50 Å) continuous layer of carbon using a Vitrobot Mark III at 4°C and 100% humidity with 30 s wait time and 3 s blot time. A dataset of 877 micrographs was collected using an automated data collection pipeline in SerialEM on a Talos Arctica operated at 200 kV. Images were acquired at a nominal magnification of 36,000x (corresponding to a pixel size of 1.169 Å) with a Gatan K2 direct electron detector in super-resolution mode and defocus values ranging from −1.3 to −3 μm. Each movie was acquired at 5 frames/s over a total exposure time of 8 s and a dose rate of 5.4 electrons/pixel/s.To take advantage of recent developments in cryo-EM image processing, we reprocessed datasets of ribosomes isolated in the presence of a dominant-negative eukaryotic release factor (DN-eRF1) . All proTo generate reference templates for auto-picking, 2000 ribosomal particles were picked manually and subjected to 2D classification. The best five classes (out of 10) were low-pass filtered to 20 Å and used for auto-picking in RELION . For autAll selected particles were subjected to an initial round of refinement using RELION’s 3D autorefine in which a 30 Å low-pass filtered map of the rabbit 80S ribosome with tRNAs but no factors (EMD-4129)  was usedTo isolate particles corresponding to different classes within the set of 80S particles we used multiple rounds of focused classification with signal subtraction (FCwSS)  centeredOnce a defined class had been isolated, the particles were realigned, and the resultant maps postprocessed in RELION. For post-processing, solvent masks were generated using relion_mask_create. Typically, these binary masks were generated from the final map from refinement low-pass filtered to 15 Å at a threshold of 0.015 or 0.02. The initial binary mask was extended by 4 Å in all directions and a raised-cosine edge was added to create a soft mask. During post-processing, phase-randomization was used to correct for the convolution effects of the solvent mask. Overall resolution estimates were calculated from Fourier shell correlations at 0.143 between the two independently refined half-maps. Final reconstructions were sharpened using automatically estimated B-factors .Processing of the human IFRD2-80S ribosomal complex was performed in RELION 2.1 as described above. After 2D classification, 22,807 particles were subject to an initial refinement using EMD-4129 low-pass filtered to 40 Å as a reference. We used the refined map, which already had clear IFRD2 density, as a reference for 3D classification to isolate 80S ribosomes, followed by a round of FCwSS centered on IFRD2 density. A final refinement of 5714 particles resulted in an 8.3 Å map without postprocessing.Our 3.6 Å map of 80S•eRF1•ABCE1 complex was used as the starting point for model building. The model of the rabbit 80S ribosome in an unrotated state (PDB accession code 5LZV)  was fittHaloarcula marismortui (PDB accession code 5Ml7) as a template. The rabbit L1 protein (NCBI ID: XP_002714675) was modeled by docking a comparative model generated using I-TASSER (Sus scrofa eEF2 (PDB accession code 3J7P) (Protein factors and tRNAs were modeled in Coot v0.8.9 . P- and I-TASSER  and morpI-TASSER . The modde 3J7P)  as a temAll models were refined using Phenix.real_space_refinement v1.13_2998 . Each roThe final models were validated using MolProbity v.4.3.1  and EMRiSoftware used in the project were installed and configured by SBGrid .Figure panels were generated using PyMOL  or Chime In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by James Manley as the Senior Editor, a Reviewing Editor and three peer reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Structures of translationally inactivated mammalian ribosomes" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The authors report cryo-EM structures of mammalian 80S ribosomes bound with SERBP1•eEF2 or with IFRD2. Importantly, they document a tRNA-binding, which differs from the canonical A, P and E sites. The structures allow near-atomic interpretation of interactions between the ribosomes, SERBP1 and eEF2, shedding new light on the mechanism of SERBP1. The work describes several new findings that will be of interest to researchers in and beyond the ribosome field.Essential revisions:The reviewers believe that your paper contains novel and interesting data, but that the paper needs revisions to (1) include reference to biochemical/biophysical work in the field (please see in the reviews below) and (2) provide a clearer explanation/review of the biological relevance of these structures as "inactivated" complexes. As you can see reviewer #1, in particular, is concerned whether the "inactive" states are just an artifact of isolation and treatment or whether they represent true inactive states that might reflect different physiological conditions. Also, there is the possibility that they represent intermediates (i.e. perhaps during recycling)?Reviewer #1:This is an interesting manuscript that reports on the structure of complexes of "inactivated" ribosomes. As such, these may offer little new insight into the process of translation but may be very important in elements relating to the regulation of translation and availability of ribosomal subunits. A major concern for this paper is whether the structures determined are biologically relevant as these inactive complexes have not been shown to directly be related to any process within the cell that might have generated such inactive particles (i.e. either as soluble complexes or as stress granules). Secondly, in the partial purification of ribosomal complexes, the authors use high speed centrifugation which is known to cause inactivation of ribosomes . Thus, there is the concern as to whether the complexes observed represent native structures or ones altered by hydrostatic pressure. However, that said, it is impressive that the authors can resolve so many structures from a large dataset and offers promise for the ability to readily obtain almost any ribosomal structure.1) Title – it is not clear that these ribosomes have been inactivated, but rather that they are in an inactive state as the A, P and E sites or mRNA channel are blocked. There is the possibility that some of these complexes (i.e. 80S•P•E•eRF1•ABCE1) may represent intermediates in the translation cycle .2) The authors use affinity tags for purification, but it is not clear that this process has actually worked. How is this population different from one where no affinity purification is used?3) It is anticipated that a comparison of the yield of different proteins as determined by mass spectrometry should be reflected in the types of particles that are examined. Is there any data that would allow one to relate the amount of protein ?4) Supplementary file 1 – Initiation factors: the authors should identify the subunits of eIF2 as α, β and γ so as to not confuse the reader with eIF2A, a separate initiation factor . Secondly, is there any explanation as to why eIF1, 1A, 5 or 5B were not found?5) IFRD2 – The authors have failed to indicate that rabbit reticulocytes are essentially "interferon treated cells" in that they have elevated levels of several normally interferon induced proteins  synthase and RNAse L). Thus, this complex is likely to not normally be present in most tissues in the absence of exposure to interferon.6) For the Z site tRNA, is it possible that this unique conformation is influenced by eIF5A which binds in this vicinity ?7) Discussion section – "tRNA binding to the Z site is more likely during periods of elevated deacylated tRNA levels.". This is not likely to occur as a slight increase in deacylated tRNA leads to the phosphorylation of eIF2 and the shutoff of protein synthesis (thus sparing the use of aminoacyl-tRNAs).Reviewer #2:Brown and co-authors describe cryo-EM structures of mammalian 80S ribosomes bound with SERBP1•eEF2  or with IFRD2. In addition, a tRNA-binding site is described, which differs from the canonical A, P and E sites. These results were obtained by re-evaluation of a previously published cryo-EM data set. The structures allow near-atomic interpretation of interactions between the ribosomes, SERBP1 and eEF2, bringing insights into the mechanism of SERBP1, however the authors' description of the mechanism is not clear (see below). The 80S•IFRD2 structure is an exciting finding, which identifies a new protein's role in translation regulation. The new tRNA position near the E site echoes a recent work on the bacterial ribosome. The authors propose that this position is on the pathway of deacyl-tRNA dissociation from the E site. Overall this work describes several new findings that will be of interest to researchers in and beyond the ribosome field.The following changes/clarifications are suggested:1) The mechanistic implications of SERBP1•eEF2 complex remain unclear. Based on the finding of SERBP1 bound together with eEF2, the authors propose that SERBP1 depletes cellular eEF2. First, it would be helpful to expand the discussion and mention that yeast homologue Stm1 binds yeast ribosomes without eEF2. Do the mechanisms of translation regulation by Stm1 and SERBP1 differ in that the latter depletes not only the 80S but also eEF2? Alternatively, the authors may have missed 80S•SERBP1 particles in their data set. Such particles may even be more abundant than 80S•SERBP1•eEF2 but also more dynamic, thus difficult to average and classify. Second, the mechanistic description of how SERBP1 prevents eEF2 dissociation remains vague. Comparison with other available eEF2 structures (with sordarin or other drugs and/or GDPNP/GDP nucleotides) might bring insights into how the conformational dynamics of eEF2 and/or the ribosome are affected by SERBP1.2) IFRD2 is proposed to regulate translation by binding to non-translating 80S. But could IFRD2 also bind vacant 40S and prevent translation initiation?3) Describe the rotational state of the 80S bound with P-tRNA and Z-tRNA . Does IFRD2 induce a different 80S conformation?Reviewer #3:This manuscript describes the results of a refined analysis of a data set of nearly 1 million ribosome particles obtained from an in vitro rabbit reticulocyte translation reaction in which a dominant negative release factor had been added to enrich for translation termination complexes . Evidently, individual complexes only represent a small portion (5-25%) of the ribosomes isolated by the procedures used and the authors have mined this reaction for complexes other than those that have already been published to define 9 new structures . There is little information in the present manuscript that describes how the complexes examined here were specifically obtained from the reticulocyte lysate, but it would appear that they work under conditions in which only 80S particles form and that the isolated 80S peak is filled with a diverse array of distinct ribosome complexes. In the Brown et al., 2015 paper it appears to describe a FLAG-tag pull down of the nascent peptide, where focus was given to the eRF1-trapped sub-population of complexes. One of the 9 complexes defined here appears to be the same structure as that which was published in Brown et al., 2015b (the eRF1-ABCE1 POST complex), indicating a duplication or positive control of sorts. While I might be able to understand how the other P-site tRNA-containing complexes were isolated by a nascent polypeptide-directed pull-down approach, it is unclear how the complexes lacking P-site tRNA were obtained (i.e. the focus of the present investigations). I would strongly suggest further clarification on these points for the sake of reproducibility.The two translationally inactive complexes discussed in detail in Figure 2, Figure 3 and Figure 4 are certainly interesting in regards to the mechanisms by which cells may "store" excess ribosomes and the quality of the data appear sufficient enough to support the claims that the authors make, which are largely qualitative descriptions of the SERBP1 and IFRD2 binding sites and the global positioning of the deacylated tRNA within the E site. In this regard, I think the paper is perfectly suitable for publication.out) described by , I don't see how they make this connection. I also don't understand the authors reference to the "known role [of the L1 stalk] in escorting deacylated tRNA from the ribosome", citing two bodies of work in bacteria that make no such claim or data to support such a claim. In this regard, is it really wise to give it a name other than E' or something of the sort? I fear that re-naming it the "Z" site will significantly complicate its description in future publications. The new position the authors see is clearly an "E site" position, yes?What appears to need further clarification and elaboration is the discussion surrounding the "Z" site for tRNA binding. Alternative positions for E-site tRNA have been described since the early 80s in the early fluorescence studies of eukaryotic ribosomes . Movements of deacylated tRNA within the E site have also been specifically described in recent single-molecule fluorescence studies . Although the authors state that their "Z" site position may be similar or related to the position .Although SERBP1/Stm1 has been implicated in stress responses , inactive ribosomes may also occur outside periods of stress. Both SERBP1 and IFRD2 are expressed in most tissues at moderate levels (https://www.proteinatlas.org/ENSG00000214706-IFRD2/tissue and https://www.proteinatlas.org/ENSG00000142864-SERBP1/tissue) and are detected to be associated with ribosomes in unstressed cells . This suggests that a percentage of ribosomes exist in inactivate states even in cells not undergoing stress. As we mention in the paper, one possibility is that ribosomes are inactivated as part of translational reprogramming that occurs during differentiation. As our ribosomes were purified from reticulocytes, the penultimate stage of erythrocyte differentiation, this may explain the presence of inactive ribosomes in our sample. Consistent with this, we observe higher IFRD2 levels in reticulocytes than in HEK293T cells .Secondly, in the partial purification of ribosomal complexes, the authors use high speed centrifugation which is known to cause inactivation of ribosomes . Thus, there is the concern as to whether the complexes observed represent native structures or ones altered by hydrostatic pressure. However, that said, it is impressive that the authors can resolve so many structures from a large dataset and offers promise for the ability to readily obtain almost any ribosomal structure.Though we cannot completely rule out the possibility that high-speed centrifugation may inactivate some reticulocyte ribosomes, we believe there is strong evidence that the structures of inactive ribosomes we analyze represent physiological complexes and are not the result of technical artifacts:First, the ability to detect known intermediates of translation  demonstrates that our purification scheme is capable to retaining ribosomes in near-native states. In addition, using the same centrifugation conditions as in this manuscript, we can isolate ribosomal pellets that translate just as efficiently in fractionated cell-free translation systems as complete reticulocyte lysate , suggesting that these centrifugation conditions do not significantly inactivate ribosomes. Similar centrifugation conditions are used to isolate ribosomes to generate other prokaryotic and eukaryotic cell-free translation systems , including the translation system used in Lu et al., 1997. We think this is consistent with the studies by Lu et al., 1997 and Scheck and Landau, 1982, which primarily analyze ribosome activity after applying high-pressure treatments in closed pressure vessels, rather than by centrifugation.Second, the interaction between IFRD2 and ribosomes is also detected in a mass spectrometry study that did not use high-speed centrifugation , as well as in immunoprecipitations of Flag-tagged IFRD2 from 293T cell lysate that was not subject to centrifugation . We also detect endogenous IFRD2 quantitatively associated with ribosomes in reticulocyte lysate before  and after  highspeed centrifugation. Finally, recombinant IFRD2 only weakly associates with ribosomes after centrifugation when incubated with lysate , unlike 1) Title – it is not clear that these ribosomes have been inactivated, but rather that they are in an inactive state as the A, P and E sites or mRNA channel are blocked. There is the possibility that some of these complexes (i.e. 80S•P•E•eRF1•ABCE1) may represent intermediates in the translation cycle .As shown in Figure 1 we only consider ribosomal complexes containing SERBP1 and IFRD2 to be inactive. To more accurately describe these complexes, we have changed the title to “Structures of translationally inactive mammalian ribosomes”.2) The authors use affinity tags for purification, but it is not clear that this process has actually worked. How is this population different from one where no affinity purification is used?We address this comment in detail in response to reviewer #3.3) It is anticipated that a comparison of the yield of different proteins as determined by mass spectrometry should be reflected in the types of particles that are examined. Is there any data that would allow one to relate the amount of protein ?m/z values are unequal, the peptides detected may not fully correlate with the protein composition of the sample. We do detect many more proteins by mass spectrometry (Supplementary file 1) than we identified by cryo-EM analysis (Table 1). This may be due to numerous factors, including relative protein abundance, mRNA-binding proteins that do not directly contact the ribosome, or the inability to visualize flexible ribosome-binding proteins.Our mass spectrometry analysis was run for protein detection of SDS-PAGE gel lanes and not quantification (e.g. via multiplexing of different samples for comparison). As proteins respond differently to trypsin digestion, some peptides ionize better than others, and detection efficiencies for ions with different 4) Supplementary file 1 – Initiation factors: the authors should identify the subunits of eIF2 as α, β and γ so as to not confuse the reader with eIF2A, a separate initiation factor . Secondly, is there any explanation as to why eIF1, 1A, 5 or 5B were not found?We have edited Supplementary file 1 to include the names of the eIF2 subunits alongside their Uniprot IDs. We note that eIF5B did appear in our mass spectrometry analysis . The absence of other initiation factors may be due to their absence from the sample, presence at concentrations below the detection limit, or technical limitations of the mass spectrometry experiment, particularly given the small sizes of eIF1 and 1A.5) IFRD2 – The authors have failed to indicate that rabbit reticulocytes are essentially "interferon treated cells" in that they have elevated levels of several normally interferon induced proteins  synthase and RNAse L). Thus, this complex is likely to not normally be present in most tissues in the absence of exposure to interferon.As we mention in the manuscript, the name “interferon-related developmental regulator” appears to be a misnomer that originated from a mistaken sequence alignment with mouse interferon-b . We do not know of a direct connection between IFRD2 expression or function and interferon signaling, though we cannot exclude the possibility that IFRD2, like established interferon-induced proteins, may be upregulated in certain situations to inhibit translation. In addition, IFRD2 is expressed in many tissues (https://www.proteinatlas.org/ENSG00000214706-IFRD2/tissue), and we can also detect low levels of endogenous IFRD2 in HEK293T cells . We therefore think that IFRD2 is a general mechanism of translationally repressing ribosomes.6) For the Z site tRNA, is it possible that this unique conformation is influenced by eIF5A which binds in this vicinity ?eIF5A was our first thought when we saw density in this area prior to classification. However, following extensive classification, we see no evidence of eIF5A bound to any ribosomes. eIF5A has been observed in another cryo-EM study 7) Discussion section – "tRNA binding to the Z site is more likely during periods of elevated deacylated tRNA levels.". This is not likely to occur as a slight increase in deacylated tRNA leads to the phosphorylation of eIF2 and the shutoff of protein synthesis (thus sparing the use of aminoacyl-tRNAs).Work in yeast has shown that phosphorylation of eIF2α is induced within 15 min of amino acid starvation . This means elevated concentrations of deacylated tRNA can occur even if only transiently before eIF2 phosphorylation dampens translation. We have rephrased the text to make this clearer.Reviewer #2:1) The mechanistic implications of SERBP1•eEF2 complex remain unclear. Based on the finding of SERBP1 bound together with eEF2, the authors propose that SERBP1 depletes cellular eEF2. First, it would be helpful to expand the discussion and mention that yeast homologue Stm1 binds yeast ribosomes without eEF2. Do the mechanisms of translation regulation by Stm1 and SERBP1 differ in that the latter depletes not only the 80S but also eEF2?The thinking that Stm1 binds the yeast ribosome in the absence of eEF2 is influenced by the absence of eEF2 in structures of the yeast 80S in complex with Stm1 . However, work from Nono Takeuchi-Tomita’s lab provides evidence that Stm1 stabilizes eEF2 on the yeast ribosome . It is therefore possible that Stm1 and SERBP1 function similarly within cells to deplete eEF2, but yeast eEF2 was lost during purification or did not crystallize.Alternatively, the authors may have missed 80S•SERBP1 particles in their data set. Such particles may even be more abundant than 80S•SERBP1•eEF2 but also more dynamic, thus difficult to average and classify. Second, the mechanistic description of how SERBP1 prevents eEF2 dissociation remains vague. Comparison with other available eEF2 structures (with sordarin or other drugs and/or GDPNP/GDP nucleotides) might bring insights into how the conformational dynamics of eEF2 and/or the ribosome are affected by SERBP1.We agree with the reviewer that SERBP1 probably binds ribosomes in the absence of eEF2 and cannot discount the presence of 80S•SERBP1 in our dataset. However, as eEF2 is one of the most highly expressed proteins in cells, ribosome•SERBP1 complexes may quickly recruit eEF2.The exact mechanism for how SERBP1 prevents eEF2 from dissociating from the ribosome is difficult to infer from our two structural snapshots. However, it is clear that SERBP1 does not prevent GTP hydrolysis or induce large displacements in orientations of the domains of eEF2. Rather, its mechanism of action may be similar to sordarin, an antibiotic that also does not prevent GTP hydrolysis or induce large structural changes yet still traps eEF2 on the ribosome. Sordarin binds in a pocket between domains III, IV and V and in doing so increases interdomain contacts and subtly constrain the domains in conformations that prevent release . As SERBP1 interacts only with domain IV of eEF2, it is likely that SERBP1 constrains the interdomain movements necessary for release by locking domain IV in a fixed position in the A site. We have altered the text to include a more detailed proposal for how we expect SERBP1 to prevent eEF2 dissociation.2) IFRD2 is proposed to regulate translation by binding to non-translating 80S. But could IFRD2 also bind vacant 40S and prevent translation initiation?IFRD2 interacts with both the small and large subunits, suggesting that the interaction is most stable with 80S ribosomes. Additionally, our immunoprecipitation experiments with FLAG-tagged IFRD2 did not isolate any 40S•IFRD2 complexes . Despite these observations, we cannot preclude the possibility that IFRD2 binds vacant 40S subunits before recruiting 60S subunits and in some cases a tRNA bound at the Z site. Regardless of the assembly pathway, IFRD2 would function to repress translation.3) Describe the rotational state of the 80S bound with P-tRNA and Z-tRNA . Does IFRD2 induce a different 80S conformation?The ribosome is in a non-rotated state in the presence of IFRD2 and/or Z-site tRNA. We have added this information to the manuscript.Reviewer #3:[…] There is little information in the present manuscript that describes how the complexes examined here were specifically obtained from the reticulocyte lysate, but it would appear that they work under conditions in which only 80S particles form and that the isolated 80S peak is filled with a diverse array of distinct ribosome complexes. In the Brown et al., 2015 paper it appears to describe a FLAG-tag pull down of the nascent peptide, where focus was given to the eRF1-trapped sub-population of complexes. One of the 9 complexes defined here appears to be the same structure as that which was published in Brown et al., 2015b (the eRF1-ABCE1 POST complex), indicating a duplication or positive control of sorts. While I might be able to understand how the other P-site tRNA-containing complexes were isolated by a nascent polypeptide-directed pull-down approach, it is unclear how the complexes lacking P-site tRNA were obtained (i.e. the focus of the present investigations). I would strongly suggest further clarification on these points for the sake of reproducibility.The original aim of the purification strategy was to isolate 80S•P•E•eRF1•ABCE1 complexes using a FLAG-tagged nascent chain and a catalytically inactive mutant of eRF1 in cell-free translational reactions. The complexes lacking peptidyl-tRNA are likely ribosomes that either co-associate with the target ribosome-nascent-chain complexes or bind non-specifically to the anti-FLAG beads during purification. This apparent co-association and/or non-specific binding also occurs in our experiments with FLAG-tagged IFRD2, where 75% of immunoprecipitated ribosomes observed by cryo-EM lack IFRD2. The phenomenon of achieving enrichment rather than homogeneity is widely reported with many target cryo-EM structures coming from about 10% of the total data, for example the ribosomal complexes described in Voorhees and Hegde, 2015; Shen et al., 2015; Braunger et al., 2018.The two translationally inactive complexes discussed in detail in Figure 2, Figure 3 and Figure 4 are certainly interesting in regards to the mechanisms by which cells may "store" excess ribosomes and the quality of the data appear sufficient enough to support the claims that the authors make, which are largely qualitative descriptions of the SERBP1 and IFRD2 binding sites and the global positioning of the deacylated tRNA within the E site. In this regard, I think the paper is perfectly suitable for publication.What appears to need further clarification and elaboration is the discussion surrounding the "Z" site for tRNA binding. Alternative positions for E-site tRNA have been described since the early 80s in the early fluorescence studies of eukaryotic ribosomes . Movements of deacylated tRNA within the E site have also been specifically described in recent single-molecule fluorescence studies . Although the authors state that their "Z" site position may be similar or related to the position (Eout) described , I don't see how they make this connection. I also don't understand the authors reference to the "known role [of the L1 stalk] in escorting deacylated tRNA from the ribosome", citing two bodies of work in bacteria that make no such claim or data to support such a claim. In this regard, is it really wise to give it a name other than E' or something of the sort? I fear that re-naming it the "Z" site will significantly complicate its description in future publications. The new position the authors see is clearly an "E site" position, yes?Aside from these major points, the paper certainly seems publishable in its present format from my perspective.We decided to call the site as the “Z site” for a number of reasons. Firstly, the naming of E-site intermediates in bacteria is confusing and we did not want to add to this confusion. Published terms include Eout , E2 , E´ , and F site . We felt a different letter would help distinguish the site from reported intermediates in bacterial ribosomes. Secondly, using E´ (or similar) may give the impression that this tRNA position is definitely an intermediate of ejection from the E site. Although this is a plausible explanation, we cannot ignore the possibility that a tRNA can only bind to the Z site when the normal passage of tRNAs through the ribosome is stopped. Thirdly, “Z” as the last letter of the alphabet evokes the extreme position on the ribosome adopted by the tRNA.The comparison with Eout  was based on the way in which the anti-codon stem-loop of the tRNAs in both complexes interact with the extremity of the small subunit. However, given that the Z-site is specific to eukaryotes, we have removed this comparison. We have also removed the references to the role of the L1 in escorting deacylated tRNAs, except to say that the L1 stalk interacts with all known tRNA ejection intermediates.