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      High-resolution structure of a Shigella type III secretion needle by solid-state NMR and cryo-electron microscopy

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          We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state NMR (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-Å cryo-EM density map and 996 ssNMR distance constraints, the structure of the Type-III Secretion System (T3SS) needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent dataset of 691 ssNMR constraints and STEM measurements. The hybrid model resolves the conformation of the non-conserved N-terminus, that occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.

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          Most cited references 52

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          ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules.

          We have recently completed a full re-architecturing of the ROSETTA molecular modeling program, generalizing and expanding its existing functionality. The new architecture enables the rapid prototyping of novel protocols by providing easy-to-use interfaces to powerful tools for molecular modeling. The source code of this rearchitecturing has been released as ROSETTA3 and is freely available for academic use. At the time of its release, it contained 470,000 lines of code. Counting currently unpublished protocols at the time of this writing, the source includes 1,285,000 lines. Its rapid growth is a testament to its ease of use. This chapter describes the requirements for our new architecture, justifies the design decisions, sketches out central classes, and highlights a few of the common tasks that the new software can perform. © 2011 Elsevier Inc. All rights reserved.
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            Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.

            Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra. CANDID uses an iterative approach with multiple cycles of NOE cross-peak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers. The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra. The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle. CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e. network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle. Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE cross-peak assignments. Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation. CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates. The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins. The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis. The fact that the correct fold is obtained in cycle 1 of a de novo structure calculation is the single most important advance achieved with CANDID, when compared with previously proposed automated NOESY assignment methods that do not use network-anchoring and constraint-combination.
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              An improved broadband decoupling sequence for liquid crystals and solids.

              Recently we developed an efficient broadband decoupling sequence called SPARC-16 for liquid crystals ¿J. Magn. Reson. 130, 317 (1998). The sequence is based upon a 16-step phase cycling of the 2-step TPPM decoupling method for solids ¿J. Chem. Phys. 103, 6951 (1995). Since then, we have found that a stepwise variation of the phase angle in the TPPM sequence offers even better results. The application of this new method to a liquid crystalline compound, 4-n-pentyl-4'-cyanobiphenyl, and a solid, L-tyrosine hydrochloride, is reported. The reason for the improvement is explained by an analysis of the problem in the rotating frame. Copyright 2000 Academic Press.

                Author and article information

                Nat Commun
                Nat Commun
                Nature communications
                15 August 2014
                29 September 2014
                29 March 2015
                : 5
                : 4976
                [1 ]Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany
                [2 ]Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germany
                [3 ]Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
                [4 ]Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA
                Author notes
                [* ]Corresponding authors: alange@ (A.Lange) and sgourn@ (N.G.S.)

                Equally contributing authors.

                Current address: Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), Institut Européen de Chimie et Biologie (IECB), CNRS - Universite Bordeaux - Institut Polytechnique Bordeaux, All. Geoffroy Saint-Hilaire 33600 Pessac, France (A.L.) and Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA (N.G.S.)


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