Chiminey: Reliable Computing and Data Management Platform in the Cloud

The enabling of scientific experiments that are embarrassingly parallel, long running and data-intensive into a cloud-based execution environment is a desirable, though complex undertaking for many researchers. The management of such virtual environments is cumbersome and not necessarily within the core skill set for scientists and engineers. We present here Chiminey, a software platform that enables researchers to (i) run applications on both traditional high-performance computing and cloud-based computing infrastructures, (ii) handle failure during execution, (iii) curate and visualise execution outputs, (iv) share such data with collaborators or the public, and (v) search for publicly available data.Comment: Preprint, ICSE 201

The Alliance for Cellular Signaling Plasmid Collection: A Flexible Resource for Protein Localization Studies and Signaling Pathway Analysis

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines

Epitope Flexibility and Dynamic Footprint Revealed by Molecular Dynamics of a pMHC-TCR Complex

The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent ‘snapshots ’ and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on a2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6–9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into ‘scanning ’ mechanism of SB27 that extends the role of the germline encoded CDR2a and CDR2b loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB2

The flexibility of the LPEP peptide within HLA-B*3508 while complexed to the SB27 TCR.

<p>(A) RMSFs as calculated from MD simulations for MHC and peptide residues. Only positions 1 to 180 of the MHC molecules are displayed with the MHC helices underlined. HLA-B*3508-LPEP simulations 1 and 2 are in red and orange respectively and HLA-B*3508-LPEP SB27 TCR simulations 1 and 2 are in dark purple and pink respectively. The boxed insert highlights the RMSFs of the peptide at positions 1 to 13; (B) Cartoon representation of the crystal structure of HLA-B*3508-LPEP SB27 TCR complex (PDB ID 2AK4) with helices coloured grey, peptide in yellow and polymorphic residue in green. For clarity the SB27 TCR has been removed. MD snapshots of the peptide taken every 10 ns are colour coded as per A.</p

MHC and peptide conformational flexibility in MD simulations.

<p>(A) RMSFs as calculated from MD simulations for MHC and peptide residues (inset). Only positions 1 to 180 of the MHC molecules are displayed with the MHC helices underlined. HLA-B*3508 simulations 1 and 2 are in red and orange respectively and HLA-B*3501 simulations 1 and 2 are in dark and light blue respectively. The boxed inset highlights the RMSFs of the peptide at positions 1 to 13; (B) Cartoon representation of the crystal structure of HLA-B*3508-LPEP (PDB ID 1ZHL) with helices coloured grey, peptide in yellow and polymorphic residue in green. MD snapshots of the peptide taken every 10 ns and colour coded in red for simulation 1 and orange for simulation 2; (C) Cartoon representation of the crystal structure of HLA-B*3501-LPEP (PDB ID 1ZHK) with helices shown in grey, peptide in yellow and polymorphic residue in green. MD snapshots taken each 10 ns are colour coded dark blue for simulation 1 and light blue for simulation 2.</p

TCR-pMHC scanning.

<p>The crystal structure of (A) complex A and (B) complex B is in grey for reference and the maximum movement of the TCR during the respective simulations is superposed in light blue or dark blue. Orthogonal orientations are shown in the right hand panels. The arrow highlights the direction of TCR movement during the respective simulations.</p

Hydrogen bonds between the pMHC and the TCR observed over the course of the simulations.

<p>(-) indicates no interaction observed in simulation.</p

Overall cartoon of TCR-pMHC complex SB27-HLA-B*3508-LPEP (PDB ID 2AK4).

<p>(A) HLA-B*3508 shown in grey with polymorphic residue (R156) in green, LPEP peptide shown in red, SB27 TCR shown in yellow and cyan for α- and β- chains respectively; (B) Superposition of the two distinct TCR-pMHC complexes within the crystal structure, SB27 α- and β-chains of complex A coloured orange and dark blue respectively and complex B coloured as per (A); (C) aerial view of antigen binding cleft of HLA-B*3508-LPEP with colours as per (A) and the SB27 TCR removed with the exception of the CDR loops.</p

The SB27 TCR footprint on HLA-B*3508-LPEP following MD simulation.

<p>(A) A comparison of contacts observed in both the crystal structure and dynamics. MHC or peptide contacts observed in both the crystal structure and dynamics coloured red and orange respectively. Residues coloured in light or dark blue represent novel short-lived (&lt;50%) or long-lived (&gt;50%) interactions respectively that were observed only during the simulation. The footprint represents both complex A and complex B as all contacts were conserved between the two simulations with the exception of Arg157 which was a short-lived contact only observed in the simulation of complex A and Arg151 and Arg162 which were long-lived contacts only observed in the simulation of complex A; (B) The HLA-B*3508 and LPEP residues contacted by the TCR are coloured according to the CDR loop from which they are derived. The uncontacted regions of LPEP are in red, residues contacted by CDR2α in yellow, CDR3α in cyan, CDR1β in green and CDR2β in purple.</p