Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Transcription factor 21 accelerates vascular calcification in mice by activating the IL-6/STAT3 signaling pathway and the interplay between VSMCs and ECs.

Vascular calcification is caused by the deposition of calcium salts in the intimal or tunica media layer of the aorta, which increases the risk of cardiovascular events and all-cause mortality. However, the mechanisms underlying vascular calcification are not fully clarified. Recently it has been shown that transcription factor 21 (TCF21) is highly expressed in human and mouse atherosclerotic plaques. In this study we investigated the role of TCF21 in vascular calcification and the underlying mechanisms. In carotid artery atherosclerotic plaques collected from 6 patients, we found that TCF21 expression was upregulated in calcific areas. We further demonstrated TCF21 expression was increased in an in vitro vascular smooth muscle cell (VSMC) osteogenesis model. TCF21 overexpression promoted osteogenic differentiation of VSMC, whereas TCF21 knockdown in VSMC attenuated the calcification. Similar results were observed in ex vivo mouse thoracic aorta rings. Previous reports showed that TCF21 bound to myocardin (MYOCD) to inhibit the transcriptional activity of serum response factor (SRF)-MYOCD complex. We found that SRF overexpression significantly attenuated TCF21-induced VSMC and aortic ring calcification. Overexpression of SRF, but not MYOCD, reversed TCF21-inhibited expression of contractile genes SMA and SM22. More importantly, under high inorganic phosphate (3 mM) condition, SRF overexpression reduced TCF21-induced expression of calcification-related genes (BMP2 and RUNX2) as well as vascular calcification. Moreover, TCF21 overexpression enhanced IL-6 expression and downstream STAT3 activation to facilitate vascular calcification. Both LPS and STAT3 could induce TCF21 expression, suggesting that the inflammation and TCF21 might form a positive feedback loop to amplify the activation of IL-6/STAT3 signaling pathway. On the other hand, TCF21 induced production of inflammatory cytokines IL-1ß and IL-6 in endothelial cells (ECs) to promote VSMC osteogenesis. In EC-specific TCF21 knockout (TCF21ECKO) mice, VD3 and nicotine-induced vascular calcification was significantly reduced. Our results suggest that TCF21 aggravates vascular calcification by activating IL-6/STAT3 signaling and interplay between VSMC and EC, which provides new insights into the pathogenesis of vascular calcification. TCF21 enhances vascular calcification by activating the IL-6-STAT3 signaling pathway. TCF21 inhibition may be a new potential therapeutic strategy for the prevention and treatment of vascular calcification.

Overexpression of NgBR inhibits high-fat diet-induced atherosclerosis in ApoE-deficiency mice.

BACKGROUND: Hyperlipidemia (hypercholesterolemia and/or hypertriglyceridemia) is a risk factor for atherosclerosis. Nogo-B receptor (NgBR) plays important roles in hepatic steatosis and cholesterol transport. However, the effect of NgBR overexpression on atherosclerosis remains unknown. MATERIALS AND METHODS: Apolipoprotein E deficient (ApoE-/-) mice infected with adeno-associated virus (AAV)-NgBR expression vector were fed a high-fat diet for 12 weeks, followed by determination of atherosclerosis and the involved mechanisms. RESULTS: We determined that high expression of NgBR by AAV injection mainly occurs in the liver and it can substantially inhibit en face and aortic root sinus lesions. NgBR overexpression also reduced levels of inflammatory factors in the aortic root and serum, and levels of cholesterol, triglyceride, and free fatty acids in the liver and serum. Mechanistically, NgBR overexpression increased the expression of scavenger receptor type BI and the genes for bile acid synthesis, and decreased the expression of cholesterol synthesis genes by reducing sterol regulatory element-binding protein 2 maturation in the liver, thereby reducing hypercholesterolemia. In addition, NgBR overexpression activated AMP-activated protein kinase α via the Ca2+ signaling pathway, which inhibited fat synthesis and improved hypertriglyceridemia. CONCLUSIONS: Taken together, our study demonstrates that overexpression of NgBR enhanced cholesterol metabolism and inhibited cholesterol/fatty acid synthesis to reduce hyperlipidemia, and reduced vascular inflammation, thereby inhibiting atherosclerosis in ApoE-/- mice. Our study indicates that NgBR might be a potential target for atherosclerosis treatment.

Symmetric activation and modulation of the human calcium-sensing receptor.

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

The Role of Histone Protein Acetylation in Regulating Endothelial Function.

Endothelial cell (EC), consisting of the innermost cellular layer of all types of vessels, is not only a barrier composer but also performing multiple functions in physiological processes. It actively controls the vascular tone and the extravasation of water, solutes, and macromolecules; modulates circulating immune cells as well as platelet and leukocyte recruitment/adhesion and activation. In addition, EC also tightly keeps coagulation/fibrinolysis balance and plays a major role in angiogenesis. Therefore, endothelial dysfunction contributes to the pathogenesis of many diseases. Growing pieces of evidence suggest that histone protein acetylation, an epigenetic mark, is altered in ECs under different conditions, and the acetylation status change at different lysine sites on histone protein plays a key role in endothelial dysfunction and involved in hyperglycemia, hypertension, inflammatory disease, cancer and so on. In this review, we highlight the importance of histone acetylation in regulating endothelial functions and discuss the roles of histone acetylation across the transcriptional unit of protein-coding genes in ECs under different disease-related pathophysiological processes. Since histone acetylation changes are conserved and reversible, the knowledge of histone acetylation in endothelial function regulation could provide insights to develop epigenetic interventions in preventing or treating endothelial dysfunction-related diseases.

NOGOB receptor-mediated RAS signaling pathway is a target for suppressing proliferating hemangioma.

Infantile hemangioma is a vascular tumor characterized by the rapid growth of disorganized blood vessels followed by slow spontaneous involution. The underlying molecular mechanisms that regulate hemangioma proliferation and involution still are not well elucidated. Our previous studies reported that NOGOB receptor (NGBR), a transmembrane protein, is required for the translocation of prenylated RAS from the cytosol to the plasma membrane and promotes RAS activation. Here, we show that NGBR was highly expressed in the proliferating phase of infantile hemangioma, but its expression decreased in the involuting phase, suggesting that NGBR may have been involved in regulating the growth of proliferating hemangioma. Moreover, we demonstrate that NGBR knockdown in hemangioma stem cells (HemSCs) attenuated growth factor-stimulated RAS activation and diminished the migration and proliferation of HemSCs, which is consistent with the effects of RAS knockdown in HemSCs. In vivo differentiation assay further shows that NGBR knockdown inhibited blood vessel formation and adipocyte differentiation of HemSCs in immunodeficient mice. Our data suggest that NGBR served as a RAS modulator in controlling the growth and differentiation of HemSCs.

Peroxisome Proliferator-Activated Receptor-Gamma Reduces ER Stress and Inflammation via Targeting NGBR Expression.

Increased Nogo-B receptor (NGBR) expression in the liver improves insulin sensitivity by reducing endoplasmic reticulum stress (ER stress) and activating the AMPK pathway, although it remains elusive the mechanisms by which NGBR is induced. In this study, we found that PPARγ ligands (rosiglitazone or pioglitazone) increased NGBR expression in hepatic cells and HUVECs. Furthermore, promoter analysis defined two PPREs (PPARγ-responsive elements) in the promoter region of NGBR, which was further confirmed by the ChIP assay. In vivo, using liver-specific PPARγ deficient (PPARγLKO) mice, we identified the key role of PPARγ expression in pioglitazone-induced NGBR expression. Meanwhile, the basal level of ER stress and inflammation was slightly increased by NGBR knockdown. However, the inhibitory effect of rosiglitazone on inflammation was abolished while rosiglitazone-inhibited ER stress was weakened by NGBR knockdown. Taken together, these findings show that NGBR is a previously unrecognized target of PPARγ activation and plays an essential role in PPARγ-reduced ER stress and inflammation.

NIR-II window tracking of hyperglycemia induced intracerebral hemorrhage in cerebral cavernous malformation deficient mice.

Second near infrared (NIR-II) window fluorescence imaging between 1000 and 1700 nm with reduced scattering and autofluorescence and deep tissue light penetration allows early and non-invasive determination of vascular pathologies. Here, we demonstrate in vivo NIR-II imaging techniques for tracking hyperglycaemia-induced Intracerebral Hemorrhage (ICH) and Blood Brain Barrier (BBB) hyperpermeability in Cerebral Cavernous Malformation (CCM) deficient mice (CCM1+/-). We synthesised PEGylated Ag2S quantum dots (QDs) with a bright fluorescent emission peak centred at 1135 nm under an 808 nm NIR light for dynamic imaging of cerebral vasculature in mice and determined the development of ICH and BBB impairment in hyperglycaemic CCM1+/- mice. In vivo optical imaging was conducted with micro-CT (including k-mean cluster analysis) as well as in vivo permeability assays using FITC-dextran perfusion and IgG staining, respectively. The increased BBB permeability in CCM1+/- mice was further demonstrated to be associated with a high-glucose-caused decrease of CCM1 expressions. This study validates that deep-penetrating NIR-II QDs can be used for the tracking of ICH and BBB hyperpermeability in transgenic mice models of cerebral vascular anomalies.

Author Correction: Structure of human GABAB receptor in an inactive state.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure of human GABAB receptor in an inactive state.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Comparative proteomic analysis of protein methylation provides insight into the resistance of hepatocellular carcinoma to 5-fluorouracil.

Protein methylation is one of the common post-translational modifications involved in diverse biological processes including signal transduction, transcriptional regulation, DNA repairing, gene activation, gene repression, and RNA processing. Due to technique limitation, the investigation of protein methylation in cancer cells is not well achieved, which hinders our understanding of the contribution of protein methylation to drug resistance. In this study, we analyzed the methylproteomes of both 5-fluorouracil (5-Fu) resistant Bel/5-Fu cell line and its parental Bel cell line by employing SPE-SCX based label-free quantitative proteomics. We identified 313 methylation forms on 294 sites in Bel cells and 294 methylation forms on 260 sites in Bel/5-Fu cells with high localization confidence. In addition, we quantified 251 methylation forms and found that 77 methylation forms significantly changed. After normalizing with the protein abundance, the 89 methylation forms were determined with the significant changes in site stoichiometry. The sequence characteristics of these significantly changed methylation sites are different. Gene ontology analysis showed that these significantly changed methylated proteins mainly involved in the biological processes of translation and transcription. Together, our findings indicated that protein methylation occurring in hepatocellular carcinoma might play a critical role in requiring drug resistance. SIGNIFICANCE: The drug resistance acquired in cancer cells has been considered as a major challenge for the cancer treatment. Due to complexity, the molecular mechanisms are still largely unknown. Identifying the key markers will improve our understanding of the mechanisms and is crucial for the development of new therapeutic strategies to overcome resistance. To date, increasing number of proteomics and phosphoproteomics studies were reported to investigate the mechanisms of drug resistance. However, the methylproteomics studies related to drug resistance were not reported yet. Here, we performed the SPE-SCX based label-free quantitative proteomics to analyze the methylproteomes of both resistant cell line Bel/5-Fu and sensitive cell line Bel. Through the qualitative and quantitative analysis, we found that the sequence characteristics of methylation sites were evidently different between these two cell lines. The results suggested that some methyltransferases might play a crucial role in the regulation of drug resistance. We also performed the analysis of methyl-site stoichiometry by normalizing the protein abundances. It was found that 89 methylation forms were determined with the significant changes in site stoichiometry, which may contribute to the development of the Bel cells into resistant cells. Our methylproteomes dataset would be useful to reveal novel molecular mechanisms of drug resistance acquired in hepatocellular carcinoma.

Quantitative proteomic and phosphoproteomic studies reveal novel 5-fluorouracil resistant targets in hepatocellular carcinoma.

The development of chemoresistance remains the major obstacles to successful chemotherapy of hepatocellular carcinoma. The molecular mechanisms of drug resistance are complex. Identifying the key markers is crucial for development of therapeutic strategies to overcome resistance. In this study, we employed a cell-line model consisting of the 5-fluorouracil resistant Bel/5-Fu cell line and its parental Bel cell line. Using stable isotope dimethyl labeling combined with high-resolution mass spectrometry, in total, 8272 unique proteins and 22,095 phosphorylation sites with high localization confidence were identified. Our data indicated that the GnRH signaling pathway was involved in acquiring drug resistance, which has not been well elucidated. The western blotting results confirmed that the expression levels of PLCß3 and PLCß3 pS1105 in Bel/5-Fu cells were increased as compared to Bel cells. Furthermore, the protein levels of SRC and PKCδ, which could phosphorylate PLCß3 at ser1105, were higher in Bel/5-Fu cells than in Bel cells. The knockdown of SRC, PKCδ and PLCß3 increased the susceptibility of Bel/5-Fu cells to 5-Fu. Besides, the increased transcription levels of PLCß3, PKCδ and SRC were significantly associated with decreased overall survival. Together, our deep proteomic and phosphoproteomic data reveal novel therapeutic targets for attenuating 5-Fu resistance in anti-cancer therapy. SIGNIFICANCE: It was reported that many hepatocellular carcinoma patients are resistance to 5-Fu. Although some studies related to drug resistance have been reported, the underlying mechanisms were not well elucidated. Unlike many single molecular studies, we focused on the global proteome and phosphoproteome analysis of Bel and Bel5-/Fu cell line using stable isotope dimethyl labeling to identify the previously unrecognized signaling pathway for causing 5-Fu resistance. Our results showed that the phosphorylation levels of PLCß3 pS1105 and the protein levels of PLCß3, PKCδ and SRC, which are major components of GnRH signaling pathway were higher in Bel/5-Fu cells than in Bel cells. Furthermore, knockdown of PLCß3, PKCδ and SRC increased the susceptibility of Bel/5-Fu cells to 5-Fu. Overall, this is the first comprehensive proteomic and phosphoproteomic studies on 5-Fu resistant cell line Bel/5-Fu to identify the potential targets of attenuating chemoresistance in hepatocellular carcinoma.

A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVß subunits.

Inhibiting high-voltage-activated calcium channels (HVACCs; CaV1/CaV2) is therapeutic for myriad cardiovascular and neurological diseases. For particular applications, genetically-encoded HVACC blockers may enable channel inhibition with greater tissue-specificity and versatility than is achievable with small molecules. Here, we engineered a genetically-encoded HVACC inhibitor by first isolating an immunized llama nanobody (nb.F3) that binds auxiliary HVACC CaVß subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to CaVß-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (CaV-aßlator), an E3 ubiquitin ligase, ablated currents from diverse HVACCs reconstituted in HEK293 cells, and from endogenous CaV1/CaV2 channels in mammalian cardiomyocytes, dorsal root ganglion neurons, and pancreatic ß cells. In cardiomyocytes, CaV-aßlator redistributed CaV1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces CaV-aßlator as a potent genetically-encoded HVACC inhibitor, and describes a general approach that can be broadly adapted to generate versatile modulators for macro-molecular membrane protein complexes.

HDAC3 Deficiency Promotes Liver Cancer through a Defect in H3K9ac/H3K9me3 Transition.

DNA damage triggers diverse cancers, particularly hepatocellular carcinoma (HCC), but the intrinsic link between DNA damage and tumorigenesis remains unclear. Because of its role as an epigenetic and transcriptional regulator, histone deacetylase 3 (HDAC3) is essential for DNA damage control and is often aberrantly expressed in human HCC. In this study, we used individual class I HDAC member-deficient mice to demonstrate that K9 in histone H3 (H3K9), which is the critical site for the assembly of DNA damage response complexes, is exclusively targeted by HDAC3. Ablation of HDAC3 disrupted the deacetylation and consequent trimethylation of H3K9 (H3K9me3), the first step in double-strand break repair, and led to the accumulation of damaged DNA. Simultaneously, hyperacetylated H3K9 (H3K9ac) served as a transcriptional activator and enhanced multiple signaling pathways to promote tumorigenesis. Together, these results show that HDAC3 targets the H3K9ac/H3K9me3 transition to serve as a critical regulator that controls both DNA damage repair and the transcription of many tumor-related genes. Moreover, these findings provide novel insights into the link between DNA damage and transcriptional reprogramming in tumorigenesis. SIGNIFICANCE: These findings show that HDAC3 exclusively regulates H3K9ac in response to DNA damage, and loss of HDAC3 activity shifts the balance from DNA damage control to protumorigenic transcriptional activity.

Structural basis for auxiliary subunit KCTD16 regulation of the GABAB receptor.

Metabotropic GABAB receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABAB receptor complexes contain the principal subunits GABAB1 and GABAB2, which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABAB receptors and modify the kinetics of GABAB receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABAB receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABAB2 receptor. A single GABAB2 C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABAB2-KCTD16 interface disrupted both the biochemical association and functional modulation of GABAB receptors and G protein-activated inwardly rectifying K+ channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABAB receptors. Defining the binding interface of GABAB receptor and KCTD reveals a potential regulatory site for modulating GABAB-receptor function in the brain.

25-Hydroxycholesterol activates the expression of cholesterol 25-hydroxylase in an LXR-dependent mechanism.

Cholesterol 25-hydroxylase (CH25H) catalyzes the production of 25-hydroxycholesterol (25-HC), an oxysterol that can play an important role in different biological processes. However, the mechanisms regulating CH25H expression have not been fully elucidated. In this study, we determined that CH25H is highly expressed in mouse liver and peritoneal macrophages. We identified several liver X receptor (LXR) response elements (LXREs) in the human CH25H promoter. In HepG2 cells, activation of LXR by 25-HC or other oxysterols and synthetic ligands [T0901317 (T317) and GW3965] induced CH25H protein expression, which was associated with increased CH25H mRNA expression. 25-HC or T317 activated CH25H transcription in an LXRE-dependent manner. Thus, high-expressing LXRα or LXRß activated CH25H expression, and the activation was further enhanced by LXR ligands. In contrast, inhibition of LXRα/ß expression attenuated 25-HC or T317-induced CH25H expression. Deficiency of interferon γ expression reduced, but did not block, LXR ligand-induced hepatic CH25H expression. Activation of LXR also substantially induced macrophage CH25H expression. In vivo, administration of GW3965 to mice increased CH25H expression in both liver and peritoneal macrophages. Taken together, our study demonstrates that 25-HC can activate CH25H expression in an LXR-dependent manner, which may be an important mechanism to exert the biological actions of 25-HC.

Structural biology of GABAB receptor.

Metabotropic GABAB receptor is a G protein-coupled receptor (GPCR) that mediates slow and prolonged inhibitory neurotransmission in the brain. It functions as a constitutive heterodimer composed of the GABAB1 and GABAB2 subunits. Each subunit contains three domains; the extracellular Venus flytrap module, seven-helix transmembrane region and cytoplasmic tail. In recent years, the three-dimensional structures of GABAB receptor extracellular and intracellular domains have been elucidated. These structures reveal the molecular basis of ligand recognition, receptor heterodimerization and receptor activation. Here we provide a brief review of the GABAB receptor structures, with an emphasis on describing the different ligand-bound states of the receptor. We will also compare these with the known structures of related GPCRs to shed light on the molecular mechanisms of activation and regulation in the GABAB system, as well as GPCR dimers in general. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

Structural mechanism of ligand activation in human calcium-sensing receptor.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca(2+) homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca(2+) and PO4(3-) ions. Both ions are crucial for structural integrity of the receptor. While Ca(2+) ions stabilize the active state, PO4(3-) ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

Data publication with the structural biology data grid supports live analysis.

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.