Lipid-Protein Interactions in Double-Layered Two-Dimensional AQP0 Crystals

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. We describe a 1.9 Å resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic N- and C-termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We could therefore build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.Molecular and Cellular Biolog

Collaboration gets the most out of software

By centralizing many of the tasks associated with the upkeep of scientific software, SBGrid allows researchers to spend more of their time on research

Conformational Locking upon Cooperative Assembly of Notch Transcription Complexes

SummaryThe Notch intracellular domain (NICD) forms a transcriptional activation complex with the DNA-binding factor CSL and a transcriptional co-activator of the Mastermind family (MAML). The “RAM” region of NICD recruits Notch to CSL, facilitating the binding of MAML at the interface between the ankyrin (ANK) repeat domain of NICD and CSL. Here, we report the X-ray structure of a human MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon stepwise assembly of a MAML1/NICD/CSL complex using HX-MS. Association of CSL with NICD exerts remarkably little effect on the exchange kinetics of the ANK domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK repeats 2-3. These exchange patterns identify critical features contributing to the cooperative assembly of Notch transcription complexes (NTCs), highlight the importance of MAML recruitment in rigidifying the ANK domain and stabilizing its interface with CSL, and rationalize the requirement for MAML1 in driving cooperative dimerization of NTCs on paired-site DNA

A grid-enabled web service for low-resolution crystal structure refinement

The deformable elastic network (DEN) method for reciprocal-space crystallographic refinement improves crystal structures, especially at resolutions lower than 3.5 Å. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements

Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides

High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex

Adapting federated cyberinfrastructure for shared data collection facilities in structural biology

It has been difficult, historically, to manage and maintain early-stage experimental data collected by structural biologists in synchrotron facilities. This work describes a prototype system that adapts existing federated cyberinfrastructure technology and techniques to manage collected data at synchrotrons and to facilitate the efficient and secure transfer of data to the owner’s home institution

Development of the Precision Link Biobank at Boston Children’s Hospital: Challenges and Opportunities

Increasingly, biobanks are being developed to support organized collections of biological specimens and associated clinical information on broadly consented, diverse patient populations. We describe the implementation of a pediatric biobank, comprised of a fully-informed patient cohort linking specimens to phenotypic data derived from electronic health records (EHR). The Biobank was launched after multiple stakeholders’ input and implemented initially in a pilot phase before hospital-wide expansion in 2016. In-person informed consent is obtained from all participants enrolling in the Biobank and provides permission to: (1) access EHR data for research; (2) collect and use residual specimens produced as by-products of routine care; and (3) share de-identified data and specimens outside of the institution. Participants are recruited throughout the hospital, across diverse clinical settings. We have enrolled 4900 patients to date, and 41% of these have an associated blood sample for DNA processing. Current efforts are focused on aligning the Biobank with other ongoing research efforts at our institution and extending our electronic consenting system to support remote enrollment. A number of pediatric-specific challenges and opportunities is reviewed, including the need to re-consent patients when they reach 18 years of age, the ability to enroll family members accompanying patients and alignment with disease-specific research efforts at our institution and other pediatric centers to increase cohort sizes, particularly for rare diseases

Structure, function and interactions of enzyme IIA from the phosphoenolpyruvate, lactose phosphotransferase system

grantor: University of TorontoThe bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is responsible for binding, transmembrane transport and phosphorylation of numerous sugar substrates. The system is also involved in the regulation of a variety of metabolic and transcriptional processes. The PTS consists of Enzyme I, heat stable protein (HPr), as well as several sugar specific multiprotein permeases known as Enzymes II. Enzyme IIALac belongs to the lactose/cellobiose family of Enzymes II, one of four functionally as well as structurally distinct groups. The structure of 'Lactococcus lactis' Enzyme IIA, the first representative of the family to have its three-dimensional structure determined, has been solved using the single isomorphous replacement and anomalous scattering (SIRAS) method at 2.3Å resolution. No overall structural homology was observed to any PTS proteins nor to any of the proteins in the PDB. Two structures of Enzyme IIALac from 'Staphylococcus aureus' (72% sequence identity) have been subsequently determined in two different crystallographic space groups using molecular replacement at 3.2Å and 3.3Å resolution respectively. The subunits of the wildtype Enzyme IIALac in both 'L. lactis' and ' S. aureus', are related to each other by the inherent three-fold rotational symmetry and pack against each other forming a nine-helix bundle which is stabilised by a centrally located Mg++. The metal binding, which because of its location we call a cationic oligomerization site (COS), is missing in the Asp81Leu mutant. This mutant protein can still be phosphorylated by HPr, but it is deficient in relaying the phosphate group to Enzyme IIB. The structures of the mutated Enzyme IIA determined at 100K to 2.1Å and at room temperature to 2.4 Å are also described. Determination of several structures of Enzymes IIA from the lactose/cellobiose family was followed by structural interpretation yielding some surprising results. Enzyme IIALac from 'L. lactis' seems to undergo breathing motions in solution, which can at least periodically separate the subunits of the molecule. Models of the complexes between Enzyme IIALac and phosphorylated HPr as well as between Enzyme IIA Lac and Enzyme IIBCel have been constructed. The Enzyme IIALac - Enzyme IIBCel complex supports biochemical data that the trimer of Enzyme IIA does not separate into subunits following the phosphorylation event.Ph.D