pFind Studio: a computational solution for mass spectrometry-based proteomics
2017
Analytical chemistry2017. Cramer, CN et al.
Novo Nordisk AS, Global Res, Prot Engn, Novo Nordisk Pk, DK-2760 Malov, Denmark.
ABSTRACT:Mapping of disulfide bonds is, an essential part of protein characterization to ensure correct cysteine pairings. For this, mass spectrometry (MS) is the most widely used technique due to fast and accurate Characterization. However, MS-based disulfide mapping is challenged when multiple disulfide bonds are present in complicated patterns. This includes the presence of disulfide bonds in nested patterns and closely spaced cysteines. Unambiguous mapping of such disulfide bonds typically requires advanced MS approaches. In this study, we exploited in-source reduction (ISR) of disulfide bonds during the electrospray ionization process to facilitate disulfide bond assignments. We successfully developed a LC-ISR-MS/MS methodology to use as an online and fully automated partial reduction procedure. Postcolumn partial reduction by ISR. provided fast and easy identification of peptides involved in disulfide bonding from nonreduced proteolytic digests, due to the concurrent detection of disulfide-containing peptide species and their composing free, peptides. Most importantly, intermediate partially reduced species containing only a single disulfide bond were also generated, from which unambiguous assignment of individual disulfide bonds could be done in species containing closely spaced disulfide bonds. The strength of this methodology was demonstrated by complete mapping of all four disulfide bonds in lysozyme and-all 17 disulfide bonds in human serum albumin, including nested disulfide bonds and motifs of adjacent cysteine residues.
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Bioinformatics2017. Liu, Y et al.
Univ Western Ontario, Dept Comp Sci, London, ON N6A 5B7, Canada.
ABSTRACT:Motivation: Enzymatic digestion under appropriate reducing conditions followed by mass spectrometry analysis has emerged as the primary method for disulfide bond analysis. The large amount of mass spectral data collected in the mass spectrometry experiment requires effective computational approaches to automate the interpretation process. Although different approaches have been developed for such purpose, they always choose to ignore the frequently observed internal ion fragments and they lack a reasonable quality control strategy and calibrated scoring scheme for the statistical validation and ranking of the reported results. Results: In this research, we present a new computational approach, DISC (DISulfide bond Characterization), for matching an input MS/MS spectrum against the putative disulfide linkage structures hypothetically constructed from the protein database. More specifically, we consider different ion types including a variety of internal ions that frequently observed in mass spectra resulted from disulfide linked peptides, and introduce an effective two-layer scoring scheme to evaluate the significance of the matching between spectrum and structure, based on which we have also developed a useful target-decoy strategy for providing quality control and reporting false discovery rate in the final results. Systematic experiments conducted on both low-complexity and high-complexity datasets demonstrated the efficiency of our proposed method for the identification of disulfide bonds from MS/MS spectra, and showed its potential in characterizing disulfide bonds at the proteome scale instead of just a single protein.
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Nature2017. Schilbach, S et al.
Max Planck Inst Biophys Chem, Dept Mol Biol, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT:For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 angstrom and 5.8 angstrom, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.
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Nature2017. Bertram, K et al.
MPI Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT:Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 angstrom, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 angstrom from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP.
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NATURE2017. Blees, A et al.
Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue Strasse 9, Frankfurt/Main, 60438, Germany
ABSTRACT:The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptideMHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells1,2. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin3,4. The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitts lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.
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Cell2017. Bertram, K et al.
MPI Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT:Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6. U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5' splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step. The molecular organization of several B-specific proteins suggests that they are involved in negatively regulating Brr2, positioning the U6/5'ss helix, and stabilizing the B complex structure. Our results indicate significant differences between the early activation phase of human and yeast spliceosomes.
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eLife2017. Sun, Q et al.
Chinese Acad Sci, CAS Ctr Excellence Biomacromol, Inst Biophys, Key Lab RNA Biol, Beijing, Peoples R China.
ABSTRACT:Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors.
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nature structural & molecular biology2017. Barandun, J et al.
Rockefeller Univ, Lab Prot & Nucle Acid Chem, 1230 York Ave, New York, NY 10021 USA.
ABSTRACT:The small-subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small-subunit processome at an overall resolution of 3.8 angstrom, which provides an essentially complete near-atomic model of this assembly. In this nucleolar superstructure, 51 ribosome-assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry and steric hindrance, as well as protein-and RNA-mediated RNA remodeling, are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.
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Cell2017. Wan, RX et al.
Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing 100084, Peoples R China.
ABSTRACT:The disassembly of the intron lariat spliceosome (ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex from Saccharomyces cerevisiae at an average resolution of 3.5 angstrom. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 50 exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly. The DEAH family ATPase/helicase Prp43 binds Syf1 at the periphery of the spliceosome, with its RNA-binding site close to the 30 end of U6 snRNA. The C-terminal domain of Ntr1/Spp382 associates with the GTPase Snu114, and Ntr2 is anchored to Prp8 while interacting with the superhelical domain of Ntr1. These structural features suggest a plausible mechanism for the disassembly of the ILS complex.
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Autophagy2017. Zheng, JX et al.
Tsinghua Univ, Tsinghua Univ Peking Univ Joint Ctr Life Sci, Sch Life Sci, State Key Lab Membrane Biol, Beijing, Peoples R China.
ABSTRACT:PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45. We also purified the ATG2B-WDR45 complex and then performed 3-dimensional reconstruction of the complex by single-particle electron microscopy, which revealed a club-shaped heterodimer with an approximate length of 22 nm. Furthermore, we performed cross-linking mass spectrometry and identified a set of highly cross-linked intermolecular and intramolecular lysine pairs. Finally, based on the cross-linking data followed by bioinformatics and mutagenesis analysis, we determined the conserved aromatic H/YF motif in the C terminus of ATG2A and ATG2B that is crucial for complex formation.
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