Automated Structure Solution With autoSHARP

Vonrhein, Clemens; Blanc, Eric; Roversi, Pietro; Bricogne, G.

doi:10.1385/1-59745-266-1:215

Cited by 849 publications

(878 citation statements)

References 28 publications

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“…The occupancies calculated by SHELX were between 100% and 58% for the first 14 sites, followed by a drop to ,25% for additional peaks that were not considered. Zinc positions were refined using autoSHARP 54 in a multiwavelength anomalous diffraction experiment using four wavelengths (Extended Data Table 2a, b). Two Pol II-based homology models of the Pol I core were fitted to match the experimental zinc positions using the program MOLOC.…”

Section: Research Article Methodsmentioning

confidence: 99%

RNA polymerase I structure and transcription regulation

Engel

Sainsbury

Cheung

et al. 2013

Nature

200

305

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Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I fromthe yeast Saccharomyces cerevisiae at 2.8A˚ resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II. An ‘expander’ element occupies the DNA template site and stabilizes an expanded active centre cleft with an unwound bridge helix. A ‘connector’ element invades the cleft of an adjacent polymerase and stabilizes an inactive polymerase dimer. The connector and expander must detach during Pol I activation to enable transcription initiation and cleft contraction by convergent movement of the polymerase ‘core’ and ‘shelf’ modules. Conversion between an inactive expanded and an active contracted polymerase state may generally underlie transcription. Regulatory factors can modulate the core–shelf interface that includes a ‘composite’ active site for RNA chain initiation, elongation, proofreading and termination

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Section: Research Article Methodsmentioning

confidence: 99%

RNA polymerase I structure and transcription regulation

Engel

Sainsbury

Cheung

et al. 2013

Nature

200

305

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“…The heavy atom positions were determined in SHELXD 32 and refined in AutoSharp 33 . The initial phases were improved by solvent flattening with SOLOMON 34 , and four-fold non-crystallographic symmetry averaging with RESOLVE 35 , generating electron density map of sufficient quality for model building.…”

Section: Methodsmentioning

confidence: 99%

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

Lee

Guo

Zeng

et al. 2017

Nature

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TMEM175 is a lysosomal K+ channel important for maintaining the lysosomal membrane potential and pH stability1. It contains two homologous copies of a six-transmembrane (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif2–4. Present in a subset of bacteria and archaea, the prokaryotic TMEM175 contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 from Chamaesiphon minutus, CmTMEM175, whose architecture represents a completely different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swap. The highly conserved TM1 acts as the pore lining inner helix, creating an hour-glass shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the C-terminal half of TM1s form a bottle neck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter plays the central role in channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears distinct from that of the classical K+ channel family.

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“…We processed all diffraction images using the XDS suite 29 . We solved the structure of GP12-CIMCD by means of SIRAS using the native dataset and the derivative dataset at the peak wavelength in AUTOSHARP 30 . We built an initial model using RESOLVE 31 and manually completed the model using Coot 32 .…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

Schulz

Dickmanns

Urlaub

et al. 2010

Nat Struct Mol Biol

106

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Proteins fold into their functional three-dimensional structure based on the information encoded in the residue sequence 1 . In all domains of life, various strategies evolved to assist folding processes in order to prevent immediate misfolding and aggregation of the nascent polypeptide chain. In the crowded cellular environment, protein folding is often aided by molecular chaperones. Molecular chaperones differ in size, function and energy dependence; however, all have in common to bind to the unfolded state of the protein in order to facilitate or mediate assembly of the correct three-dimensional structure 2,3 . In contrast to molecular chaperones, the intramolecular chaperones (IMCs) constitute a different class of chaperones. As part of the polypeptide chain, the IMC is typically cleaved off the target protein after the folding process is completed. Two classes of IMCs can be distinguished: class I IMCs assist the protein to fold into the correct tertiary structure, whereas class II IMCs are involved in quaternary structure assembly 4 . In contrast to many molecular chaperones, no evidence for an ATP-driven cleavage reaction could be found in IMCs. An example of a class II intramolecular chaperone has been identified in viral tailspike and fiber proteins. These proteins are functionally unrelated but share a highly conserved chaperone domain at their C terminus (C-terminal intramolecular chaperone domain, CIMCD), which is cleaved at a conserved position in an autoproteolytic reaction 5 . It was shown that the covalent linkage between the CIMCD and N-terminal pre-protein is necessary for correct folding, indicating that an in trans function of the chaperone is impossible 5 . Furthermore, it could be shown that some of the chaperone domains are exchangeable between the different preproteins 5,6 . While the three-dimensional structures of the CIMCDs are as yet unknown, the crystal structure of N-terminally truncated mature endoNF shows that the homotrimeric enzyme comprises a triple β-helix involved in substrate recognition 7 . This triple β-helix and the related triple-β-spiral motif have been identified in various proteins, which often play a role as virulence factors 8 . In triple β-helices, three polypeptide chains wind around a common threefold symmetry axis, conferring an extraordinary stability to the protein. The rigid elongated shape allows triple β-helix-comprising proteins to protrude from a pathogen's surface in order to interact with flexible host cell receptors, like lipopolysaccharides 9 . However, proper assembly of triple-β-helical folds poses to be difficult in the absence of a trimerization domain 10 . Hence, most triple β-helices depend on a C-terminal extension for trimerization and correct assembly 11 .Here we present the crystal structures of two representatives of a large group of systematically, functionally and structurally similar intramolecular chaperones: the Escherichia coli phage K1F endosilidase CIMCD and the Bacillus subtilis phage GA-1 neck appendage protein CIMCD. Furthermore...

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Automated Structure Solution With autoSHARP

Cited by 849 publications

References 28 publications

RNA polymerase I structure and transcription regulation

RNA polymerase I structure and transcription regulation

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

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