Cardiolipin clustering promotes mitochondrial membrane dynamics.

Cardiolipin (CL) is a mitochondria-specific phospholipid that forms heterotypic interactions with membrane-shaping proteins and regulates the dynamic remodeling and function of mitochondria. However, the precise mechanisms through which CL influences mitochondrial morphology are not well understood. In this study, employing molecular dynamics (MD) simulations, we observed CL localize near the membrane-binding sites of the mitochondrial fusion protein Optic Atrophy 1 (OPA1). To validate these findings experimentally, we developed a bromine-labeled CL probe to enhance cryoEM contrast and characterize the structure of OPA1 assemblies bound to the CL-brominated lipid bilayers. Our images provide direct evidence of interactions between CL and two conserved motifs within the paddle domain (PD) of OPA1, which control membrane-shaping mechanisms. We further observed a decrease in membrane remodeling activity for OPA1 in lipid compositions with increasing concentrations of monolyso-cardiolipin (MLCL). Suggesting that the partial replacement of CL by MLCL accumulation, as observed in Barth syndrome-associated mutations of the tafazzin phospholipid transacylase, compromises the stability of protein-membrane interactions. Our analyses provide insights into how biological membranes regulate the mechanisms governing mitochondrial homeostasis.

Structural basis of TRPV1 modulation by endogenous bioactive lipids.

TRP ion channels are modulated by phosphoinositide lipids, but the underlying structural mechanisms remain unclear. The capsaicin- and heat-activated receptor, TRPV1, has served as a model for deciphering lipid modulation, which is relevant to understanding how pro-algesic agents enhance channel activity in the setting of inflammatory pain. Identification of a pocket within the TRPV1 transmembrane core has provided initial clues as to how phosphoinositide lipids bind to and regulate the channel. Here we show that this regulatory pocket in rat TRPV1 can accommodate diverse lipid species, including the inflammatory lipid lysophosphatidic acid, whose actions are determined by their specific modes of binding. Furthermore, we show that an empty-pocket channel lacking an endogenous phosphoinositide lipid assumes an agonist-like state, even at low temperature, substantiating the concept that phosphoinositide lipids serve as negative TRPV1 modulators whose ejection from the binding pocket is a critical step toward activation by thermal or chemical stimuli.

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

Influence of geography, stroke timing, and weather conditions on transport and workflow times: Results from a longitudinal 5-year Canadian provincial registry.

BACKGROUND: In areas with high population spread such as Saskatchewan, it can be challenging to provide timely endovascular stroke treatment (EVT) to patients living far away from comprehensive stroke centres (CSC). We assessed the association of geography, stroke timing and weather conditions on EVT workflow times and clinical outcomes in Saskatchewan. METHODS: We included patients who underwent EVT between January 2017 and December 2022 in the province of Saskatchewan, Canada. Univariable and multivariable associations of time from last known well-to-CSC arrival, CSC arrival-to-reperfusion, and 90-day modified Rankin Score (mRS) with driving distance from patient home to CSC, transport mode, outdoor temperature and stroke timing (day & time) were assessed using descriptive statistics and multivariable regression. RESULTS: Three-hundred-three patients in the province of Saskatchewan underwent EVT between January 2017 and December 2022. Distance from patient home to CSC (beta-coefficient per 10 km increase = 0.02, 95% CI: 0.01-0.03) and direct to CSC transport (beta-coefficient = -0.76, 95% CI = -1.01-[-0.51]) were associated with last known well to CSC arrival time. In-hospital stroke (beta-coefficient = 0.37, 95% CI: 0.16-0.58), direct-to-CSC transfer (beta-coefficient = 0.27, 95% CI: 0.13-0.41) and daytime stroke onset (beta-coefficient = -0.15, 95% CI: -0.28-[-0.04]) were associated with time from CSC arrival to reperfusion. No association with 90-day mRS was seen. CONCLUSION: Geographic factors and stroke timing were associated with EVT workflow times. However, no association with clinical outcomes was seen, suggesting that EVT patients living remote areas of Saskatchewan have similar benefit from EVT compared to urban areas. Every effort should be made to offer timely EVT to patients from remote areas.

Structural mechanism of mitochondrial membrane remodelling by human OPA1.

Distinct morphologies of the mitochondrial network support divergent metabolic and regulatory processes that determine cell function and fate1-3. The mechanochemical GTPase optic atrophy 1 (OPA1) influences the architecture of cristae and catalyses the fusion of the mitochondrial inner membrane4,5. Despite its fundamental importance, the molecular mechanisms by which OPA1 modulates mitochondrial morphology are unclear. Here, using a combination of cellular and structural analyses, we illuminate the molecular mechanisms that are key to OPA1-dependent membrane remodelling and fusion. Human OPA1 embeds itself into cardiolipin-containing membranes through a lipid-binding paddle domain. A conserved loop within the paddle domain inserts deeply into the bilayer, further stabilizing the interactions with cardiolipin-enriched membranes. OPA1 dimerization through the paddle domain promotes the helical assembly of a flexible OPA1 lattice on the membrane, which drives mitochondrial fusion in cells. Moreover, the membrane-bending OPA1 oligomer undergoes conformational changes that pull the membrane-inserting loop out of the outer leaflet and contribute to the mechanics of membrane remodelling. Our findings provide a structural framework for understanding how human OPA1 shapes mitochondrial morphology and show us how human disease mutations compromise OPA1 functions.

Structural basis of TRPV1 modulation by endogenous bioactive lipids.

TRP ion channels are modulated by phosphoinositide lipids, but the underlying structural mechanisms remain unclear. The capsaicin- and heat-activated receptor, TRPV1, has served as a model for deciphering lipid modulation, which is relevant to understanding how pro-algesic agents enhance channel activity in the setting of inflammatory pain. Identification of a pocket within the TRPV1 transmembrane core has provided initial clues as to how phosphoinositide lipids bind to and regulate the channel. Here we show that this regulatory pocket can accommodate diverse lipid species, including the inflammatory lipid lysophosphatidic acid (LPA), whose actions are determined by their specific modes of binding. Furthermore, we show that an 'empty pocket' channel lacking an endogenous phosphoinositide lipid assumes an agonist-like state, even at low temperature, substantiating the concept that phosphoinositide lipids serve as negative TRPV1 modulators whose ejection from the binding pocket is a critical step towards activation by thermal or chemical stimuli.

Brominated lipid probes expose structural asymmetries in constricted membranes.

Lipids in biological membranes are thought to be functionally organized, but few experimental tools can probe nanoscale membrane structure. Using brominated lipids as contrast probes for cryo-EM and a model ESCRT-III membrane-remodeling system composed of human CHMP1B and IST1, we observed leaflet-level and protein-localized structural lipid patterns within highly constricted and thinned membrane nanotubes. These nanotubes differed markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion rates and lipid compositional and conformational asymmetries. Simulations and cryo-EM imaging of brominated stearoyl-docosahexanenoyl-phosphocholine showed how a pair of phenylalanine residues scored the outer leaflet with a helical hydrophobic defect where polyunsaturated docosahexaenoyl tails accumulated at the bilayer surface. Combining cryo-EM of halogenated lipids with molecular dynamics thus enables new characterizations of the composition and structure of membranes on molecular length scales.

A structural biology community assessment of AlphaFold2 applications.

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods for protein structure predictions have reached the accuracy of experimentally determined models. Although this has been independently verified, the implementation of these methods across structural-biology applications remains to be tested. Here, we evaluate the use of AlphaFold2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modeling of interactions; and modeling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modeled when compared with homology modeling, identifying structural features rarely seen in the Protein Data Bank. AF2-based predictions of protein disorder and complexes surpass dedicated tools, and AF2 models can be used across diverse applications equally well compared with experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life-science research.

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions. These vRNPs depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.

Structure and functionality of a multimeric human COQ7:COQ9 complex.

Coenzyme Q (CoQ) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined "complex Q" metabolon. Here, we present structure-function analyses of a lipid-, substrate-, and NADH-bound complex comprising two complex Q subunits: the hydroxylase COQ7 and the lipid-binding protein COQ9. We reveal that COQ7 adopts a ferritin-like fold with a hydrophobic channel whose substrate-binding capacity is enhanced by COQ9. Using molecular dynamics, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for the CoQ intermediates to translocate from the bilayer to the proteins' lipid-binding sites. Two such tetramers assemble into a soluble octamer with a pseudo-bilayer of lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors within the membrane and coordinate subsequent synthesis steps toward producing CoQ.

A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation.

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's ß subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.

Do motor plans affect sensorimotor state estimates during temporal decision-making with crossed vs. uncrossed hands? Failure to replicate the dynamic crossed-hand effect.

Hermosillo et al. (J Neurosci 31: 10019-10022, 2011) have suggested that action planning of hand movements impacts decisions about the temporal order judgments regarding vibrotactile stimulation of the hands. Specifically, these authors reported that the crossed-hand effect, a confusion about which hand is which when held in a crossed posture, gradually reverses some 320 ms before the arms begin to move from an uncrossed to a crossed posture or vice versa, such that the crossed-hand is reversed at the time of movement onset in anticipation of the movement's end position. However, to date, no other study has attempted to replicate this dynamic crossed-hand effect. Therefore, in the present study, we conducted four experiments to revisit the question whether preparing uncrossed-to-crossed or crossed-to-uncrossed movements affects the temporo-spatial perception of tactile stimulation of the hands. We used a temporal order judgement (TOJ) task at different time stages during action planning to test whether TOJs are more difficult with crossed than uncrossed hands ("static crossed-hand effect") and, crucially, whether planning to cross or uncross the hands shows the opposite pattern of difficulties ("dynamic crossed-hand effect"). As expected, our results confirmed the static crossed-hand effect. However, the dynamic crossed-hand effect could not be replicated. In addition, we observed that participants delayed their movements with late somatosensory stimulation from the TOJ task, even when the stimulations were meaningless, suggesting that the TOJ task resulted in cross-modal distractions. Whereas the current findings are not inconsistent with a contribution of motor signals to posture perception, they cast doubt on observations that motor signals impact state estimates well before movement onset.

Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance.

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

Antibiotics treat or prevent infections by killing bacteria or slowing down their growth. A large proportion of these drugs do this by disrupting an essential piece of cellular machinery called the ribosome which the bacteria need to make proteins. However, over the course of the treatment, some bacteria may gain genetic alterations that allow them to resist the effects of the antibiotic. Antibiotic resistance is a major threat to global health, and understanding how it emerges and spreads is an important area of research. Recent studies have discovered populations of resistant bacteria carrying a gene for a protein named chloramphenicol-florfenicol resistance, or Cfr for short. Cfr inserts a small modification in to the ribosome that prevents antibiotics from inhibiting the production of proteins, making them ineffective against the infection. To date, Cfr has been found to cause resistance to eight different classes of antibiotics. Identifying which mutations enhance its activity and protect bacteria is vital for designing strategies that fight antibiotic resistance. To investigate how the gene for Cfr could mutate and make bacteria more resistant, Tsai et al. performed a laboratory technique called directed evolution, a cyclic process which mimics natural selection. Genetic changes were randomly introduced in the gene for the Cfr protein and bacteria carrying these mutations were treated with tiamulin, an antibiotic rendered ineffective by the modification Cfr introduces into the ribosome. Bacteria that survived were then selected and had more mutations inserted. By repeating this process several times, Tsai et al. identified 'super' variants of the Cfr protein that lead to greater resistance. The experiments showed that these variants boosted resistance by increasing the proportion of ribosomes that contained the protective modification. This process was facilitated by mutations that enabled higher levels of Cfr protein to accumulate in the cell. In addition, the current study allowed, for the first time, direct visualization of how the Cfr modification disrupts the effect antibiotics have on the ribosome. These findings will make it easier for clinics to look out for bacteria that carry these 'super' resistant mutations. They could also help researchers design a new generation of antibiotics that can overcome resistance caused by the Cfr protein.

Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B.

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2's dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five 'aromatic fingers'. NSs binding preserves eIF2B activity by maintaining eIF2B's conformation in its active A-State.

Multivariate Analysis of Electrophysiological Signals Reveals the Time Course of Precision Grasps Programs: Evidence for Nonhierarchical Evolution of Grasp Control.

Current understanding of the neural processes underlying human grasping suggests that grasp computations involve gradients of higher to lower level representations and, relatedly, visual to motor processes. However, it is unclear whether these processes evolve in a strictly canonical manner from higher to intermediate and to lower levels given that this knowledge importantly relies on functional imaging, which lacks temporal resolution. To examine grasping in fine temporal detail here we used multivariate EEG analysis. We asked participants to grasp objects while controlling the time at which crucial elements of grasp programs were specified. We first specified the orientation with which participants should grasp objects, and only after a delay we instructed participants about which effector to use to grasp, either the right or the left hand. We also asked participants to grasp with both hands because bimanual and left-hand grasping share intermediate-level grasp representations. We observed that grasp programs evolved in a canonical manner from visual representations, which were independent of effectors to motor representations that distinguished between effectors. However, we found that intermediate representations of effectors that partially distinguished between effectors arose after representations that distinguished among all effector types. Our results show that grasp computations do not proceed in a strictly hierarchically canonical fashion, highlighting the importance of the fine temporal resolution of EEG for a comprehensive understanding of human grasp control.SIGNIFICANCE STATEMENT A long-standing assumption of the grasp computations is that grasp representations progress from higher to lower level control in a regular, or canonical, fashion. Here, we combined EEG and multivariate pattern analysis to characterize the temporal dynamics of grasp representations while participants viewed objects and were subsequently cued to execute an unimanual or bimanual grasp. Interrogation of the temporal dynamics revealed that lower level effector representations emerged before intermediate levels of grasp representations, thereby suggesting a partially noncanonical progression from higher to lower and then to intermediate level grasp control.

Is the n-back task a measure of unstructured working memory capacity? Towards understanding its connection to other working memory tasks.

Working memory is fundamental to human cognitive functioning, and it is often measured with the n-back task. However, it is not clear whether the n-back task is a valid measure of working memory. Importantly, previous studies have found poor correlations with measures of complex span, whereas a recent study (Frost et al., 2019) showed that n-back performance was correlated with a transsaccadic memory task but dissociated from performance on the change detection task, a well-accepted measure of working memory capacity. To test whether capacity is involved in the n-back task we correlated a spatial version of the test with different versions of the change detection task. Experiment 1 introduced perceptual and cognitive disruptions to the change detection task. This impacted task performance, however, all versions of the change detection task remained highly correlated with one another whereas there was no significant correlation with the n-back task. Experiment 2 removed spatial and non-spatial context from the change detection task. This produced a correlation with n-back. Our results indicate that the n-back task is supported by faculties similar to those that support change detection, but that this commonality is hidden when contextual information is available to be exploited in a change detection task such that structured representations can form. We suggest that n-back might be a valid measure of working memory, and that the ability to exploit contextual information is an important faculty captured by some versions of the change detection task.

Ribosome-associated quality control and CAT tailing.

Translation is the set of mechanisms by which ribosomes decode genetic messages as they synthesize polypeptides of a defined amino acid sequence. While the ribosome has been honed by evolution for high-fidelity translation, errors are inevitable. Aberrant mRNAs, mRNA structure, defective ribosomes, interactions between nascent proteins and the ribosomal exit tunnel, and insufficient cellular resources, including low tRNA levels, can lead to functionally irreversible stalls. Life thus depends on quality control mechanisms that detect, disassemble and recycle stalled translation intermediates. Ribosome-associated Quality Control (RQC) recognizes aberrant ribosome states and targets their potentially toxic polypeptides for degradation. Here we review recent advances in our understanding of RQC in bacteria, fungi, and metazoans. We focus in particular on an unusual modification made to the nascent chain known as a "CAT tail", or Carboxy-terminal Alanine and Threonine tail, and the mechanisms by which ancient RQC proteins catalyze CAT-tail synthesis.

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM.

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.

Grasping of Real-World Objects Is Not Biased by Ensemble Perception.

The visual system is known to extract summary representations of visually similar objects which bias the perception of individual objects toward the ensemble average. Although vision plays a large role in guiding action, less is known about whether ensemble representation is informative for action. Motor behavior is tuned to the veridical dimensions of objects and generally considered resistant to perceptual biases. However, when the relevant grasp dimension is not available or is unconstrained, ensemble perception may be informative to behavior by providing gist information about surrounding objects. In the present study, we examined if summary representations of a surrounding ensemble display influenced grip aperture and orientation when participants reached-to-grasp a central circular target which had an explicit size but importantly no explicit orientation that the visuomotor system could selectively attend to. Maximum grip aperture and grip orientation were not biased by ensemble statistics during grasping, although participants were able to perceive and provide manual estimations of the average size and orientation of the ensemble display. Support vector machine classification of ensemble statistics achieved above-chance classification accuracy when trained on kinematic and electromyography data of the perceptual but not grasping conditions, supporting our univariate findings. These results suggest that even along unconstrained grasping dimensions, visually-guided behaviors toward real-world objects are not biased by ensemble processing.