Phase combination and cross validation in iterated density-modification calculations

Cowtan, Kevin; Main, P. C.

doi:10.1107/s090744499500761x

Cited by 264 publications

(215 citation statements)

References 9 publications

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“…1). The information gained from peptidase assays and SUMO sequence alignments led to our proposal that the residues at the P 35 , P 29 , and P 5 -P 7 positions of SUMO contribute to the substrate specificity of XopD (Figs. 1, 3, and 8).…”

Section: Substrate Specificity Of Xopd-previous Results Showing Thatmentioning

confidence: 99%

“…Location of heavy atom sites, phase calculation, and refinement were performed with the program Solve (28). Density modification was carried out with the program dm (29). An initial model was constructed automatically using the program ARP/wARP (30) XopD-(335-520)(C470A) crystals were isomorphous to the wild-type crystals; model building and refinement followed a protocol similar to that used for the wild-type structure.…”

Section: Protein Expression and Purification For Crystallizationmentioning

confidence: 99%

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Structural Analysis of Xanthomonas XopD Provides Insights into Substrate Specificity of Ubiquitin-like Protein Proteases

Chosed

Tomchick

Brautigam

et al. 2007

Journal of Biological Chemistry

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XopD (Xanthomonas outer protein D), a type III secreted effector from Xanthomonas campestris pv. vesicatoria, is a desumoylating enzyme with strict specificity for its plant small ubiquitin-like modifier (SUMO) substrates. Based on SUMO sequence alignments and peptidase assays with various plant, yeast, and mammalian SUMOs, we identified residues in SUMO that contribute to XopD/SUMO recognition. Further predictions regarding the enzyme/substrate specificity were made by solving the XopD crystal structure. By incorporating structural information with sequence alignments and enzyme assays, we were able to elucidate determinants of the rigid SUMO specificity exhibited by the Xanthomonas virulence factor XopD.The small ubiquitin-like modifier (SUMO) 2 is a member of a large family of reversible post-translational modifiers. SUMO is structurally similar to ubiquitin. Like ubiquitin, SUMO utilizes a conjugation machinery (ubiquitin-activating enzyme, ubiquitin carrier protein, and ubiquitin-protein isopeptide ligase) to modify target proteins, but sumoylated proteins are not targeted for degradation by the proteasome (1). The conjugation machinery catalyzes the formation of a covalent isopeptide bond between the C-terminal glycine residue of SUMO and the ⑀-amino group of a lysine residue in the target protein. The removal of SUMO moieties from conjugated proteins by isopeptidases regenerates pools of processed SUMOs and unmodified target proteins.

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Section: Substrate Specificity Of Xopd-previous Results Showing Thatmentioning

confidence: 99%

Section: Protein Expression and Purification For Crystallizationmentioning

confidence: 99%

Structural Analysis of Xanthomonas XopD Provides Insights into Substrate Specificity of Ubiquitin-like Protein Proteases

Chosed

Tomchick

Brautigam

et al. 2007

Journal of Biological Chemistry

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“…The remaining Se site was identified from the difference Fourier map calculated with these five Se sites by using the program SHARP (14). The initial phase was recalculated by SHARP with all six Se sites and improved by solvent-flattening using the program SOLOMON (15) and noncrystallographic symmetry (NCS) averaging using the program DM (16). The initial model was manually traced and refined semiautomatically by using the program LAFIRE (M.Y., Y. Zhou, and I.T., unpublished work) with the program CNS (17).…”

Section: Methodsmentioning

confidence: 99%

Molecular basis of alanine discrimination in editing site

Sokabe

Okada

Yao

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA Ala . Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid L-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the ␤-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans-and cis-editing of tRNA Ala .alanyl-tRNA synthetase ͉ class II tRNA synthetase ͉ crystal structure ͉ trans-editing A minoacyl-tRNA synthetases (aaRSs) establish the genetic code through aminoacylation of cognate tRNA (1). However, in some aaRSs, the affinity difference of the active site is not large enough to distinguish among similar amino acids with sufficient accuracy. Therefore, during evolution, an additional editing domain that specifically hydrolyzes mischarged tRNAs has assembled with the catalytic domain to comprise contemporary aaRSs (2). In accordance with this model, the genes that autonomously encode an editing domain were distributed in many organisms (3-5), and, indeed, some of them are shown to be responsible for the transediting activity of mischarged tRNAs (4, 5). AlaX is the one such protein that shows homology to the class II alanyl-tRNA synthetase (AlaRS) editing domain ( Fig. 1) and is widely scattered among all three kingdoms of life (3). The specific activities of the archaeal AlaXs from Methanosarcina barkeri and Sulfolobus solfataricus have recently been shown to specifically hydrolyze mischarged Ser-and Gly-tRNA Ala in vitro (4).In contrast to the well established class I aaRSs, information regarding editing of the evolutionarily distinct class II aaRSs (to which AlaRS belongs) has only recently begun to emerge. The editing mechanism of the threonyl system has been investigated by structural analyses of the bacterial threonyl-tRNA synthetase (ThrRS) editing domain (hereafter called ThrRS-N2) complexed with the serine product or with the substrate analogue seryl-3Ј-aminoadenosine (SerA76) (6). These analyses showed that (i) cognate threonine and noncognate serine are discriminated by steric exclusion of the additional ␥-methyl group of threonine by the conserved His-77, Tyr-104, and Asp-180, and (ii) the HXXXH and CXXXH motifs characteristic of ThrRS-N2 should not bind a zinc ion for catalysis, despite the capability to bind zinc (7). The AlaX͞AlaRS editing domains are evolutionarily related to ThrRS-N2 and share the characteristic HXXXH and CXXXH motifs (Fig. 1) (4, 8), which were also shown to be important for the deacylation activity of AlaRS (9). The inclusion of non...

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“…Briefly, the expected heavy atoms were determined by SHELXD (Schneider and Sheldrick, 2002), and then initial phases were determined using Phaser (McCoy et al, 2007). Density modification was performed by DM (Cowtan and Main, 1996). Approximately 90% of the Dare-PTX residues were traced automatically by ARP/wARP (Perrakis et al, 2001).…”

Section: Data Refinement and Structure Determinationmentioning

confidence: 99%

Crystal structures for short-chain pentraxin from zebrafish demonstrate a cyclic trimer with new recognition and effector faces

Chen

Yuan

et al. 2015

Journal of Structural Biology

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Phase combination and cross validation in iterated density-modification calculations

Cited by 264 publications

References 9 publications

Structural Analysis of Xanthomonas XopD Provides Insights into Substrate Specificity of Ubiquitin-like Protein Proteases

Structural Analysis of Xanthomonas XopD Provides Insights into Substrate Specificity of Ubiquitin-like Protein Proteases

Molecular basis of alanine discrimination in editing site

Crystal structures for short-chain pentraxin from zebrafish demonstrate a cyclic trimer with new recognition and effector faces

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