The 1.1 Å resolution structure of a periplasmic phosphate-binding protein fromStenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) fromAlcaligenessp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement usingMOLREPin which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in theMOLREPpeak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. ABLASTsearch then indicated that the molecule was most probably a phosphate-binding protein fromStenotrophomonas maltophilia(UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of theS. maltophiliaprotein has been refined to anRfactor of 10.15% and anRfreeof 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles.

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Preliminary time-of-flight neutron diffraction studies ofEscherichia coliABC transport receptor phosphate-binding protein at the Protein Crystallography Station

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14009704 ◽

2014 ◽

Vol 70 (6) ◽

pp. 819-822 ◽

Cited By ~ 3

Author(s):

K. H. Sippel ◽

J. Bacik ◽

F. A. Quiocho ◽

S. Z. Fisher

Keyword(s):

Neutron Diffraction ◽

Binding Protein ◽

Protein Crystallography ◽

Phosphate Transport ◽

Phosphate Binding ◽

Science Center ◽

Data Set ◽

Acceptor Group ◽

Phosphate Binding Protein ◽

Ph Range

Inorganic phosphate is an essential molecule for all known life. Organisms have developed many mechanisms to ensure an adequate supply, even in low-phosphate conditions. In prokaryotes phosphate transport is instigated by the phosphate-binding protein (PBP), the initial receptor for the ATP-binding cassette (ABC) phosphate transporter. In the crystal structure of the PBP–phosphate complex, the phosphate is completely desolvated and sequestered in a deep cleft and is bound by 13 hydrogen bonds: 12 to protein NH and OH donor groups and one to a carboxylate acceptor group. The carboxylate plays a key recognition role by accepting a phosphate hydrogen. PBP phosphate affinity is relatively consistent across a broad pH range, indicating the capacity to bind monobasic (H2PO4−) and dibasic (HPO42−) phosphate; however, the mechanism by which it might accommodate the second hydrogen of monobasic phosphate is unclear. To answer this question, neutron diffraction studies were initiated. Large single crystals with a volume of 8 mm3were grown and subjected to hydrogen/deuterium exchange. A 2.5 Å resolution data set was collected on the Protein Crystallography Station at the Los Alamos Neutron Science Center. Initial refinement of the neutron data shows significant nuclear density, and refinement is ongoing. This is the first report of a neutron study from this superfamily.

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Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444999000827 ◽

1999 ◽

Vol 55 (4) ◽

pp. 826-834 ◽

Cited By ~ 22

Author(s):

Shannon W. N. Au ◽

Claire E. Naylor ◽

Sheila Gover ◽

Lucy Vandeputte-Rutten ◽

Deborah A. Scopes ◽

...

Keyword(s):

Crystal Form ◽

Search Model ◽

Molecular Replacement ◽

Asymmetric Unit ◽

Data Set ◽

Space Groups ◽

Successful Search ◽

Using Data ◽

Glucose 6 Phosphate Dehydrogenase ◽

Resolution Data

Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement. Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively. A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 Å resolution data set and a dimer of the large β + α domains of the Leuconostoc mesenteroides enzyme as a search model. This tetramer was the only successful search model for the P212121 crystal form using data to 3 Å. Crystals of the deletion mutant ΔG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 Å resolution data have been collected. Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.

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Determination of crystal structures of proteins of unknown identity using a marathon molecular replacement procedure: structure of Stenotrophomonas maltophilia phosphate-binding protein

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316012419 ◽

2016 ◽

Vol 72 (10) ◽

pp. 1081-1089 ◽

Cited By ~ 7

Author(s):

Kaushik Hatti ◽

Ashutosh Gulati ◽

Narayanaswamy Srinivasan ◽

M. R. N. Murthy

Keyword(s):

Electron Density ◽

Stenotrophomonas Maltophilia ◽

Large Scale ◽

Binding Protein ◽

Data Sets ◽

Molecular Replacement ◽

Phosphate Binding ◽

Replacement Method ◽

Replacement Procedure ◽

Phosphate Binding Protein

During the past decade, the authors have collected a few X-ray diffraction data sets from protein crystals that appeared to be easy cases of molecular replacement but failed to yield structures even after extensive trials. Here, the use of a large-scale molecular replacement method that explores all structurally characterized domains as phasing models to determine the structure corresponding to two data sets collected at 1.9 and 2.3 Å resolution is reported. These two structures were of the same protein independently crystallized in 2007 and 2011. The structures derived are virtually identical and were found to consist of two compact globular domains connected by a hinge. The high resolution of one of these data sets enabled inference of the amino-acid sequence from the electron-density map. The deduced sequence is nearly identical to that of a protein from the multidrug-resistant bacterium Stenotrophomonas maltophilia. Although the structure of this protein has not been determined previously, it is homologous to the well studied DING proteins which mediate the cellular uptake of phosphate ions. The final electron-density maps from both of the data sets revealed a large density at the interface of the two globular domains that is likely to represent a phosphate ion. Thus, the structure is likely to be that of a phosphate-binding protein encoded by the S. maltophilia genome (SmPBP; PDB entry 5j1d). The nature of the phosphate-binding site of SmPBP closely resembles that of Pseudomonas fluorescens DING (PfluDING), which displays remarkable discrimination between the closely similar phosphate and arsenate ions. The results presented here illustrate that routine crystallization trials may occasionally lead to the serendipitous crystallization of a protein of unknown identity and brute-force molecular replacement through `fold space' might allow the identification of the unknown protein.

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SIMBAD: a sequence-independent molecular-replacement pipeline

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318005752 ◽

2018 ◽

Vol 74 (7) ◽

pp. 595-605 ◽

Cited By ~ 12

Author(s):

Adam J. Simpkin ◽

Felix Simkovic ◽

Jens M. H. Thomas ◽

Martin Savko ◽

Andrey Lebedev ◽

...

Keyword(s):

Sequence Similarity ◽

Model Identification ◽

Data Bank ◽

Crystal Form ◽

Lattice Parameter ◽

Search Model ◽

Molecular Replacement ◽

Brute Force ◽

Search Models ◽

Mistaken Identity

The conventional approach to finding structurally similar search models for use in molecular replacement (MR) is to use the sequence of the target to search against those of a set of known structures. Sequence similarity often correlates with structure similarity. Given sufficient similarity, a known structure correctly positioned in the target cell by the MR process can provide an approximation to the unknown phases of the target. An alternative approach to identifying homologous structures suitable for MR is to exploit the measured data directly, comparing the lattice parameters or the experimentally derived structure-factor amplitudes with those of known structures. Here, SIMBAD, a new sequence-independent MR pipeline which implements these approaches, is presented. SIMBAD can identify cases of contaminant crystallization and other mishaps such as mistaken identity (swapped crystallization trays), as well as solving unsequenced targets and providing a brute-force approach where sequence-dependent search-model identification may be nontrivial, for example because of conformational diversity among identifiable homologues. The program implements a three-step pipeline to efficiently identify a suitable search model in a database of known structures. The first step performs a lattice-parameter search against the entire Protein Data Bank (PDB), rapidly determining whether or not a homologue exists in the same crystal form. The second step is designed to screen the target data for the presence of a crystallized contaminant, a not uncommon occurrence in macromolecular crystallography. Solving structures with MR in such cases can remain problematic for many years, since the search models, which are assumed to be similar to the structure of interest, are not necessarily related to the structures that have actually crystallized. To cater for this eventuality, SIMBAD rapidly screens the data against a database of known contaminant structures. Where the first two steps fail to yield a solution, a final step in SIMBAD can be invoked to perform a brute-force search of a nonredundant PDB database provided by the MoRDa MR software. Through early-access usage of SIMBAD, this approach has solved novel cases that have otherwise proved difficult to solve.

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Crystallization and x-ray diffraction studies of a phosphate-binding protein involved in active transport in Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)57501-9 ◽

1986 ◽

Vol 261 (17) ◽

pp. 7995-7996

Author(s):

B D Kubena ◽

H Luecke ◽

H Rosenberg ◽

F A Quiocho

Keyword(s):

Escherichia Coli ◽

Active Transport ◽

Binding Protein ◽

X Ray Diffraction ◽

Phosphate Binding ◽

X Ray ◽

Phosphate Binding Protein

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Purification and properties of glucose 1-phosphate binding protein in Agrobacterium tumefaciens

Biochimica et Biophysica Acta (BBA) - Protein Structure ◽

10.1016/0005-2795(73)90337-1 ◽

1973 ◽

Vol 328 (1) ◽

pp. 114-123

Author(s):

Sakuzo Fukui ◽

Kazuko Isobe

Keyword(s):

Agrobacterium Tumefaciens ◽

Binding Protein ◽

Phosphate Binding ◽

Purification And Properties ◽

Phosphate Binding Protein

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The high-affinity phosphate-binding protein PstS is accumulated under high fructose concentrations and mutation of the corresponding gene affects differentiation in Streptomyces lividans

Microbiology ◽

10.1099/mic.0.27983-0 ◽

2005 ◽

Vol 151 (8) ◽

pp. 2583-2592 ◽

Cited By ~ 42

Author(s):

Margarita Díaz ◽

Ana Esteban ◽

José Manuel Fernández-Abalos ◽

Ramón I. Santamaría

Keyword(s):

Culture Media ◽

Binding Protein ◽

Protein Pattern ◽

Phosphate Transport ◽

Streptomyces Lividans ◽

Null Mutant ◽

Phosphate Binding ◽

Genetic Switch ◽

High Affinity ◽

Phosphate Binding Protein

The secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Δppk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.

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