Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus.

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.

Malaria parasite translocon structure and mechanism of effector export.

The putative Plasmodium translocon of exported proteins (PTEX) is essential for transport of malarial effector proteins across a parasite-encasing vacuolar membrane into host erythrocytes, but the mechanism of this process remains unknown. Here we show that PTEX is a bona fide translocon by determining structures of the PTEX core complex at near-atomic resolution using cryo-electron microscopy. We isolated the endogenous PTEX core complex containing EXP2, PTEX150 and HSP101 from Plasmodium falciparum in the 'engaged' and 'resetting' states of endogenous cargo translocation using epitope tags inserted using the CRISPR-Cas9 system. In the structures, EXP2 and PTEX150 interdigitate to form a static, funnel-shaped pseudo-seven-fold-symmetric protein-conducting channel spanning the vacuolar membrane. The spiral-shaped AAA+ HSP101 hexamer is tethered above this funnel, and undergoes pronounced compaction that allows three of six tyrosine-bearing pore loops lining the HSP101 channel to dissociate from the cargo, resetting the translocon for the next threading cycle. Our work reveals the mechanism of P. falciparum effector export, and will inform structure-based design of drugs targeting this unique translocon.

Native structure of the RhopH complex, a key determinant of malaria parasite nutrient acquisition.

The RhopH complex is implicated in malaria parasites' ability to invade and create new permeability pathways in host erythrocytes, but its mechanisms remain poorly understood. Here, we enrich the endogenous RhopH complex in a native soluble form, comprising RhopH2, CLAG3.1, and RhopH3, directly from parasite cell lysates and determine its atomic structure using cryo-electron microscopy (cryo-EM), mass spectrometry, and the cryoID program. CLAG3.1 is positioned between RhopH2 and RhopH3, which both share substantial binding interfaces with CLAG3.1 but make minimal contacts with each other. The forces stabilizing individual subunits include 13 intramolecular disulfide bonds. Notably, CLAG3.1 residues 1210 to 1223, previously predicted to constitute a transmembrane helix, are embedded within a helical bundle formed by residues 979 to 1289 near the C terminus of CLAG3.1. Buried in the core of the RhopH complex and largely shielded from solvent, insertion of this putative transmembrane helix into the erythrocyte membrane would likely require a large conformational rearrangement. Given the unusually high disulfide content of the complex, it is possible that such a rearrangement could be initiated by the breakage of allosteric disulfide bonds, potentially triggered by interactions at the erythrocyte membrane. This first direct observation of an exported Plasmodium falciparum transmembrane protein-in a soluble, trafficking state and with atomic details of buried putative membrane-insertion helices-offers insights into the assembly and trafficking of RhopH and other parasite-derived complexes to the erythrocyte membrane. Our study demonstrates the potential the endogenous structural proteomics approach holds for elucidating the molecular mechanisms of hard-to-isolate complexes in their native, functional forms.

Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu.

X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (cryoEM) maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria-causing parasite Plasmodium falciparum, an organism that has resisted conventional structural-biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.

Post-transplant ejection fraction and renal failure in cardiac transplant recipients: An analysis of the OPTN database.

BACKGROUND: Despite improved life expectancy from a heart transplant, transplant recipients remain at high risk for renal dysfunction and failure, including end-stage kidney disease (ESKD). The onset of ESKD is a poor prognostic marker and is associated with increased mortality in this setting, as in others. There is a need to identify risk factors for ESKD among heart transplant recipients in contemporary settings. METHODS: We conducted an analysis of adult heart transplant recipients transplanted between 2008 and 2021 in the Organ Procurement and Transplantation Network database. 22 737 adult recipients of heart transplants alone were included in this analysis. We examined LVEF measured 1 year after transplant, and LVEF updated annually for association with ESKD using multivariate Cox regression models. RESULTS: LVEF at 1-year after transplant was associated with ESKD in multivariate models (Hazard Ratio 1.33 per 10-unit decrease, 95% CI 1.23-1.43, p < .001). In multivariate models using categorized LVEF, mildly reduced ejection fraction (EF 40%-50%) was associated with ESKD (HR 1.76, 95% CI 1.45-2.14, p < .001), as was reduced ejection fraction (EF < 40%, HR 2.86, 95% CI 2.01-4.07, p < .001), relative to individuals with preserved ejection fraction (EF > 50%). These associations were consistent when using annually updated ejection fraction. CONCLUSIONS: Post-transplant left ventricular ejection fraction has value in predicting end stage kidney disease among adults who receive heart transplants alone. LVEF is routinely measured as part of contemporary post heart transplant care, and a diminished LVEF should signal to clinicians that a recipient is at increased risk of renal failure.

Care and Complexity in Emergency Housing: an Examination of the COVID-19 Shelter-in-Place (SIP) Hotel Program to House People Experiencing Homelessness in San Francisco.

In this study, we consider the patient, provider, and public health repercussions of San Francisco's (SF) COVID-related response to homelessness using tourist hotels to house people experiencing homelessness (PEH). We describe the demographics, medical comorbidities, and healthcare utilization patterns of a subset of PEH who accessed the shelter-in-place (SIP) hotel sites during the 2020-2021 pandemic. We focus on how SIP hotels impacted connection to outpatient care and higher-cost emergency utilization. Our mixed methods study integrates qualitative and quantitative data to consider the impact of this temporary housing initiative among a medically complex cohort in a time of increased morbidity and mortality related to substance use. We found that temporary SIP housing increased outpatient care and reduced higher-cost hospital utilization. Our results can inform the future design and implementation of integrated supportive housing models to reduce mortality and promote wellness for PEH.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded ß-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

Building atomic models based on near atomic resolution cryoEM maps with existing tools.

The EMDataBank Validation Challenge was a challenging task for students newly introduced to the cryoEM and molecular modeling fields. However, the competition provided an effective space for student modelers to discover and explore the potentials of atomic modeling and refinement by practicing on published atomic structures. Here, by employing manual molecular modeling programs such as Coot, Phenix, and Chimera, we have regularized and improved three targets. The T20S proteasome and TRPV1 ion channel allowed us to broaden our understanding of these modeling techniques while the 70S ribosome served as a challenge to test the limits of our abilities. We were successful in our efforts to improve each of the models and provide here our cohesive methodology for de novo modeling with and without homology models, which may serve as a starting point for other undergraduates and researchers just entering the realm of cryoEM. Additionally, we provide some constructive criticism to facilitate the introduction of said undergraduates and researchers into cryoEM in the future.

Investigating the Association between Steatotic Liver Disease and CKD in a Nationally Representative Sample.

BACKGROUND: Steatotic liver disease (SLD) and chronic kidney disease (CKD) are common conditions that are strongly associated. Yet, there is a paucity of data regarding the prevalence of this overlap and the factors that may drive its occurrence. METHODS: Using the National Health and Nutrition Examination survey, we examined trends among adult participants from 2005 - 2020 that defined SLD with the Fatty Liver Index. We completed correlative analyses among adult participants from 2017 - 2020 that defined SLD based on FibroScan results. We utilized multivariable survey-weighted binomial generalized linear models to determine the factors that were associated with CKD, defined as eGFR <60 or urine albumin-creatinine-ratio >30. RESULTS: Among the 76,496 participants included in the trend analyses, the estimated prevalence of CKD was 15.7% (95%CI 15.2 - 16.2%) and SLD was 42.3% (95%CI 41.4 - 43.2%). As compared to those without SLD, those with SLD had a significantly higher estimated prevalence of CKD (SLD: 15.7%, 95%CI 14.9 - 16.5% v. No SLD 11.2%, 95%CI 10.7 - 11.7%). In multivariate analyses of 3,667 participants who underwent FibroScan and had SLD by Fatty Liver Index, adjusting for control and presence of DM, HTN, and HLD, compared to those with normal liver stiffness, those with moderate scarring (F2) had similar odds of CKD (1.53, 95CI 0.91-2.56), those with severe scarring (F3) had higher odds of CKD (2.28, 95CI 1.20-4.32), and those with cirrhosis had higher odds of CKD (2.21, 95CI 1.13-4.32). CONCLUSIONS: Our findings highlight that CKD is common among patients with SLD and that higher degrees of hepatic fibrosis are associated with CKD, independent of other co-morbidities of the metabolic syndrome.

Association of urine biomarkers of kidney health with subclinical cardiovascular disease among men with and without HIV.

OBJECTIVE: The aim of this study was to determine whether urine biomarkers of kidney health are associated with subclinical cardiovascular disease among men with and without HIV. DESIGN: A cross-sectional study within the Multicenter AIDS Cohort Study (MACS) among 504 men with and without HIV infection who underwent cardiac computed tomography scans and had urine biomarkers measured within the preceding 2 years. METHODS: Our primary predictors were four urine biomarkers of endothelial (albuminuria), proximal tubule dysfunction (alpha-1-microglobulin [A1 M] and injury (kidney injury molecule-1 [KIM-1]) and tubulointerstitial fibrosis (pro-collagen-III N-terminal peptide [PIIINP]). These were evaluated for association with coronary artery calcium (CAC) prevalence, CAC extent, total plaque score, and total segment stenosis using multivariable regression. RESULTS: Of the 504 participants, 384 were men with HIV (MWH) and 120 were men without HIV. In models adjusted for sociodemographic factors, cardiovascular disease risk factors, eGFR, and HIV-related factors, each two-fold higher concentration of albuminuria was associated with a greater extent of CAC (1.35-fold higher, 95% confidence interval 1.11-1.65), and segment stenosis (1.08-fold greater, 95% confidence interval 1.01-1.16). Associations were similar between MWH and men without HIV in stratified analyses. The third quartile of A1 M showed an association with greater CAC extent, total plaque score, and total segment stenosis, compared with the lowest quartile. CONCLUSION: Worse endothelial and proximal tubule dysfunction, as reflected by higher urine albumin and A1 M, were associated with greater CAC extent and coronary artery stenosis.

Nonselective beta-blockers may lead to stage 2 acute kidney injury and waitlist mortality in child class C cirrhosis.

BACKGROUND AND AIMS: Nonselective beta-blockers (NSBB) protect patients with compensated cirrhosis; however, it is unclear if NSBB is associated with acute kidney injury (AKI) in patients with decompensated cirrhosis. We aimed to determine if the use of NSBB was associated with an increased risk of stage II AKI or greater and waitlist mortality (WLM) among patients with decompensated cirrhosis awaiting liver transplant stratified by cirrhosis severity. METHODS: Included were 1816 outpatients listed for liver transplantation at UCSF from June 2012 to April 2022. Our primary outcome was stage 2 AKI (>200% increase in serum creatinine). Our secondary outcome was WLM (all-cause mortality). Our primary exposure was the use of any NSBB derived using natural language processing of clinical notes. Multivariable Cox proportional hazards models with time-dependent variables were used to determine the HR of NSBB use on stage 2 AKI and WLM, stratified by Child-Pugh Score. RESULTS: The average age of the cohort was 58 years old, with 35% identifying as female. In multivariable time-dependent models, NSBB use was associated with 1.53 × (95 CI 1.19-1.97) the hazard of stage 2 AKI in the cohort overall and 1.80 × (95 CI 1.26-2.57) among those with Child C cirrhosis, respectively. Similarly, NSBB use was associated with 1.30 × (95 CI 1.07-1.59) and 1.45 × (95 CI 1.03-2.03) the hazard of WLM, overall and in Child C, respectively. NSBB use was not significantly associated with AKI nor WLM among those with Child A. CONCLUSION: NSBB use is associated with Stage 2 AKI and WLM in patients awaiting liver transplantation and Child C cirrhosis. These data suggest cautious use of NSBBs in patients in this population.

Risk Factors for Arrhythmic Death, Overall Mortality, and Ventricular Tachyarrhythmias Requiring Shock After Myocardial Infarction.

The VEST (Vest Prevention of Early Sudden Death Trial) showed a trend toward decreased sudden death and lower overall mortality with a wearable cardioverter-defibrillator (WCD) in the postmyocardial infarction (post-MI) period. However, it is unclear which patients should receive WCD therapy. We aimed to identify the risk factors for arrhythmic death, all-cause mortality, and ventricular tachyarrhythmias requiring appropriate shock to identify patients most likely to benefit from a WCD. The VEST trial included patients with acute MI with ejection fraction ≤35%. Using logistic regression, 7 risk factors were evaluated for association with arrhythmic death, all-cause mortality, and appropriate shock. Among 2,302 participants, 44 had arrhythmic death (1.9%) and 86 died of any cause (3.7%). Among 1,524 participants randomized to WCD, 20 experienced appropriate shock (1.3%) over 90 days. In the multivariable analyses, lower systolic blood pressure (SBP; odds ratio [OR] 1.64 per 10 mm Hg) and higher heart rate at discharge (OR 1.19 per 10 beats/min) were associated with arrhythmic death. Lower SBP (OR 1.37) and higher heart rate (OR 1.10) were associated with all-cause mortality. Higher heart rate (OR 1.20) was associated with appropriate shock. Patients with both SBP ≤100 and heart rate ≥100 were at increased odds of arrhythmic death (OR 4.82), all-cause mortality (OR 3.10), and appropriate shock (OR 6.13). In patients with acute MI and reduced ejection fraction, lower SBP and higher heart rate at discharge were strongly associated with arrhythmic death and all-cause mortality. In conclusion, these risk factors identify a select group at high risk of adverse events in a setting where WCD therapy is reasonable.

Ambulatory urine biomarkers associations with acute kidney injury and hospitalization in people with HIV.

BACKGROUND: People with HIV (PWH) generally have worse ambulatory levels of kidney injury biomarkers and excess risk of acute kidney injury (AKI) compared to persons without HIV. We evaluated whether ambulatory measures of subclinical kidney injury among PWH are associated with subsequent AKI. METHODS: In the Predictors of Acute Renal Injury Study (PARIS), which enrolled 468 PWH from April 2016 to August 2019, we measured 10 urine biomarkers of kidney health (albumin, a1m, b2M, NGAL, IL18, KIM-1, EGF, UMOD, MCP-1, YKL40) at baseline and annually during follow-up. Using multivariable Cox regression models, we evaluated baseline and time-updated biomarker associations with the primary outcome of AKI (≥0.3 mg/dl or ≥1.5-times increase in serum creatinine from baseline) and secondary outcome of all-cause hospitalization. RESULTS: At baseline, the mean age was 53 years old, and 45% self-identified as female. In time-updated models adjusting for sociodemographic factors, comorbidities, albuminuria, estimated glomerular filtration rate, and HIV-associated factors, higher KIM-1 [hazard ratio (HR) = 1.30 per twofold higher; 95% confidence interval (CI) 1.03-1.63] and NGAL concentrations (HR = 1.24, 95% CI 1.06-1.44) were associated with higher risk of hospitalized AKI. Additionally, in multivariable, time-updated models, higher levels of KIM-1 (HR = 1.19, 95% CI 1.00, 1.41), NGAL (HR = 1.13, 95% CI 1.01-1.26), and MCP-1 (HR = 1.20, 95% CI 1.00, 1.45) were associated with higher risk of hospitalization. CONCLUSIONS: Urine biomarkers of kidney tubular injury, such as KIM-1 and NGAL, are strongly associated with AKI among PWH, and may hold potential for risk stratification of future AKI.

Structured Moderate Exercise and Biomarkers of Kidney Health in Sedentary Older Adults: The Lifestyle Interventions and Independence for Elders Randomized Clinical Trial.

Rationale & Objective: In the Lifestyle Interventions and Independence for Elders (LIFE) trial, a structured exercise intervention slowed kidney function decline in sedentary older adults. Biomarkers of kidney health could distinguish potential mechanisms for this beneficial effect. Study Design: Randomized controlled trial. Setting & Population: A total of 1,381 sedentary adults aged 70-89 years enrolled in the LIFE trial. Intervention: Structured, 2-year, moderate-intensity exercise intervention versus health education. Outcomes: Physical activity was measured by step count. Primary outcomes were changes in 14 serum and urine biomarkers of kidney health collected at baseline, year 1, and year 2. We determined the effect of randomization on changes in kidney measures and then evaluated observational associations of achieved activity on each measure. Results: Participants assigned to exercise walked on average 291 more steps per day than participants assigned to health education. The intervention was not significantly associated with changes in biomarkers of kidney health. In observational analyses, persons in the highest versus lowest quartile of activity (≥3,470 vs <1,568 steps/day) had significant improvement in urine albumin (mean, -0.22 mg albumin/g urine creatinine [interquartile range (IQR), -0.37 to -0.06]), alpha-1-microglobulin (-0.18 mg/L [-0.28 to -0.08]), trefoil factor-3 (-0.24 pg/mL [-0.35 to -0.13]), epidermal growth factor (0.19 pg/mL [0.06-0.32]), uromodulin (0.06 pg/mL [0.00-0.12]), interleukin 18 (-0.09 pg/mL [-0.15 to -0.03]), neutrophil gelatinase-associated lipocalin (-0.16 pg/mL [-0.24 to -0.07]), monocyte chemoattractant protein-1 (-0.25 pg/mL [-0.36 to -0.14]), clusterin (-0.16 pg/mL [-0.30 to -0.02]), serum tumor necrosis factor receptor-1 (-0.25 mg/dL [-0.39 to -0.11]) and tumor necrosis factor receptor-2 (-0.30 mg/dL [-0.44 to -0.16]). In sensitivity analyses, incremental changes in activity were most impactful on urine interleukin 18 and serum tumor necrosis factor-1. Limitations: The original study was not designed to assess the impact on kidney health. Non-white individuals and patients with advanced chronic kidney disease are underrepresented. Conclusions: Randomization to structured exercise did not improve kidney health at a group level. However, higher exercise was associated with concurrent improvements in biomarkers of glomerular injury, tubular function/repair, tubular injury, generalized inflammation, and tubulointerstitial repair/fibrosis. Plain-Language Summary: In the Lifestyle Interventions For Elders (LIFE) study, randomization to an exercise and physical activity intervention improved the slope of estimated glomerular filtration rate over 2 years compared with health education among older adults. In this study, we sought to determine whether there were specific biomarkers of kidney health that were affected by the exercise and physical activity intervention to investigate potential mechanisms for this positive impact on kidney decline. We found that randomization to the intervention did not improve any of the 14 measures of kidney tubule health. However, in observational analyses, higher activity was independently associated with improvements in several domains, especially tubular injury and generalized inflammation. These results help to clarify the impact of physical activity on kidney health.

Pretransplant Dialysis and Preemptive Transplant in Living Donor Kidney Recipients.

Background: The optimal timing of dialysis access placement in individuals with stage 5 CKD is challenging to estimate. Preemptive living donor kidney transplant (LDKT) is the gold-standard treatment for ESKD due to superior graft survival and mortality, but dialysis initiation is often required. Among LDKT recipients, we sought to determine which clinical characteristics were associated with preemptive transplant. Among non-preemptive LDKT recipients, we sought to determine what dialysis access was used, and their duration of use before receipt of living donor transplant. Methods: We retrospectively extracted data on 569 LDKT recipients, >18 years old, who were transplanted between January 2014 and July 2019 at UCSF, including dialysis access type (arteriovenous fistula [AVF], arteriovenous graft [AVG], peritoneal dialysis catheter [PD], and venous catheter), duration of dialysis, and clinical characteristics. Results: Preemptive LDKT recipients constituted 30% of our cohort and were older, more likely to be White, more likely to have ESKD from polycystic kidney disease, and less likely to have ESKD from type 2 diabetes. Of the non-preemptive patients, 26% used AVF, 0.5% used AVG, 32% used peritoneal catheter, 11% used venous catheter, and 31% used more than one access type. Median (IQR) time on dialysis for AVF/AVG use was 1.86 (0.85-3.32) years; for PD catheters, 1.12 (0.55-1.92) years; for venous catheters, 0.66 (0.23-1.69) years; and for multimodal access, 2.15 (1.37-3.72) years. Conclusions: We characterized the dialysis access landscape in LDKT recipients. Venous catheter and PD were the most popular modality in the first quartile of dialysis, and patients using these modalities had shorter times on dialysis compared with those with an AVF. Venous catheter or PD can be considered a viable bridge therapy in patients with living donor availability given their shorter waitlist times. Earlier referral of patients with living donor prospects might further minimize dialysis need.

Atomic structure of the translation regulatory protein NS1 of bluetongue virus.

Bluetongue virus (BTV) non-structural protein 1 (NS1) regulates viral protein synthesis and exists as tubular and non-tubular forms in infected cells, but how tubules assemble and how protein synthesis is regulated are unknown. Here, we report near-atomic resolution structures of two NS1 tubular forms determined by cryo-electron microscopy. The two tubular forms are different helical assemblies of the same NS1 monomer, consisting of an amino-terminal foot, a head and body domains connected to an extended carboxy-terminal arm, which wraps atop the head domain of another NS1 subunit through hydrophobic interactions. Deletion of the C terminus prevents tubule formation but not viral replication, suggesting an active non-tubular form. Two zinc-finger-like motifs are present in each NS1 monomer, and tubules are disrupted by divalent cation chelation and restored by cation addition, including Zn2+, suggesting a regulatory role of divalent cations in tubule formation. In vitro luciferase assays show that the NS1 non-tubular form upregulates BTV mRNA translation, whereas zinc-finger disruption decreases viral mRNA translation, tubule formation and virus replication, confirming a functional role for the zinc-fingers. Thus, the non-tubular form of NS1 is sufficient for viral protein synthesis and infectious virus replication, and the regulatory mechanism involved operates through divalent cation-dependent conversion between the non-tubular and tubular forms.

Solution Structures of Engineered Vault Particles.

Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (∼4.8 Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10 Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault.

Response to Comment on "Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing".

Wang et al question whether Lamin B receptor is required for Xist-mediated silencing because they claim that our cells contain an inversion rather than a deletion. We present evidence that these cells contain a proper deletion and that the confusion is caused by DNA probes used in the experiment. Accordingly, the points raised have no effect on the conclusions in our paper.

Structures and stabilization of kinetoplastid-specific split rRNAs revealed by comparing leishmanial and human ribosomes.

The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome.