Automated Structure Solution with the PHENIX Suite

Zwart, Peter H.; Afonine, Pavel V.; Grosse‐Kunstleve, Ralf W.; Hung, Li‐Wei; Ioerger, Thomas R.; McCoy, Airlie J.; McKee, Erik; Moriarty, Nigel W.; Read, R.J.; Sacchettini, James C.; Sauter, Nicholas K.; Storoni, Laurent C.; Terwilliger, Thomas C.; Adams, Paul D.

doi:10.1007/978-1-60327-058-8_28

Cited by 507 publications

(518 citation statements)

References 26 publications

Supporting

Mentioning

515

Contrasting

Unclassified

Order By: Relevance

“…54). The model was subjected to iterative rounds of rebuilding into 2F o À F c and F o À F c electron density maps using COOT 55 and refined using the phenix.refine subroutine from the PHENIX programme suite 56 . Data collection, refinement and model statistics are summarized in Table 1.…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

et al. 2014

View full text Add to dashboard Cite

Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH-and temperature-dependent pore-forming activity of LLO, which is unique among cholesteroldependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca 2 þ oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca 2 þ uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.

show abstract

Section: Discussionmentioning

confidence: 99%

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

et al. 2014

View full text Add to dashboard Cite

show abstract

“…We built an initial model using RESOLVE 31 and manually completed the model using Coot 32 . As we obtained the native dataset from a merohedrally twinned crystal (apparent spacegroup P6), we refined the structure using the native dataset in spacegroup P3, applying the respective twin-operator in PHENIX [33][34][35] . We determined the structure of pre-cleavage endoNF-CIMCD by molecular replacement using the β-helix domain of endoNF , PDB 1V0E 7 and the structure of GP12-CIMCD as a search model in MOLREP 36 and PHASER 37 .…”

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

View full text Add to dashboard Cite

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...

show abstract

“…During the intervening time, approximately half of the volume of the reservoir solution had evaporated, leaving a much higher ($2 M) concentration of ammonium sulfate. By comparing the unit-cell parameters of the different crystal categories (A, B, C and D), we found that the decrease in length of the cubic unit-cell axis from 185 Å in crystal category D to 167 Å in crystal category (a) Stereo pair of an electron-density map (1.0 ) calculated with the starting experimental phases after density improvement with AUTOSOLVE (Zwart et al, 2008). The manually placed three-helix bundle is shown as a yellow tube model; the threefold crystallographic axis is indicated by a red line.…”

Section: Wbdd Crystals Vary In Diffraction Qualitymentioning

confidence: 99%

“…The anomalous signal was found to extend to about 4.5 Å resolution using SHELXC (Sheldrick, 2008) and phenix.xtriage (Zwart et al, 2008) (Fig. 2), and five of the expected seven selenium sites were located by SHELXD (Sheldrick, 2008).…”

Section: Solving the Selenium Substructure And Determining The Correcmentioning

confidence: 99%

Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase fromEscherichia coliO9a

Hagelueken

Huang

Harlos

et al. 2012

Acta Crystallogr D Biol Cryst

View full text Add to dashboard Cite

WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å ) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å ) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations.

show abstract

Automated Structure Solution with the PHENIX Suite

Cited by 507 publications

References 26 publications

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

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

Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase fromEscherichia coliO9a

Contact Info

Product

Resources

About