It’s not clear whether the drop well surface can induce heterogeneous nucleation to increase the probability of screening success, although a medium-throughput 48-well SBS (the Society for Biomolecular Screening, USA) polystyrene microplate has been widely used for protein crystallization screening. Here, our accumulating data suggested that this 48-well SBS microplate with rough surface is beneficial for protein crystallization. It was observed that some protein crystals were only formed on the rough edges and/or walls of the drop well, but not on the flat bottom or in the crystallization drop. It was further demonstrated with two standard test proteins. The protuberances, grooves and cavities with varied sizes and shapes at micrometer-scale in the rough drop well wall can serve as nucleants and induce heterogeneous nucleation of protein crystals. The rough wall microplate provides a potent tool for protein crystallographers embarking on crystallization trials.

Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.

Allostery is well-documented for proteins but less recognized for DNA-protein interactions, in which DNA has been often considered as a mere template providing recognition sequences. Here we report that for two proteins bound on DNA at a separation of tens of base pairs, their DNA binding affinities can be significantly altered. This coupling effect oscillates between positive and negative cooperativity, depending on the separation distance between the two proteins on the DNA. With a DNA hairpin experiment, we provide definitive evidence for the structural basis of DNA allostery. We prove this effect is not due to protein-protein interactions but originates from the distortion of the inter-helical distance along the linker DNA. The oscillation has a periodicity of ∼10 base pairs, the helical pitch of the B-form DNA, and a characteristic decay length of ∼15 base pairs. In the theoretical analysis, we elucidate the relation between the mechanical structural distortion of DNA induced by protein-binding and the free energy coupling measured thermodynamically, providing a complete picture for the origin of DNA allostery. The allosteric coupling between two DNA-bound proteins is found to be ubiquitous, regardless of proteins' properties, implying its general roles in gene regulation. We demonstrate such DNA allostery affects gene expression levels in live E.coli cells. Pertinent to eukaryotic gene expression, we show that the binding affinity of a transcription factor depends on its separation from nearby nucleosomes. This work provides the first comprehensive study of allostery through DNA, with the understanding of its physical underpinning and ubiquity and biological relevance.

Allostery is well documented for proteins but less recognized for DNA-protein interactions. Here, we report that specific binding of a protein on DNA is substantially stabilized or destabilized by another protein bound nearby. The ternary complex's free energy oscillates as a function of the separation between the two proteins with a periodicity of ~10 base pairs, the helical pitch of B-form DNA, and a decay length of ~15 base pairs. The binding affinity of a protein near a DNA hairpin is similarly dependent on their separation, which-together with molecular dynamics simulations-suggests that deformation of the double-helical structure is the origin of DNA allostery. The physiological relevance of this phenomenon is illustrated by its effect on gene expression in live bacteria and on a transcription factor's affinity near nucleosomes.

Despite impressive advances in theories, methods and technologies, crystallization still remains a serious bottleneck in structural determination of macromolecules. Here we present a novel solid–liquid interface method (SLIM) for protein crystallization, based on the pre-adding and drying of a crystallization reagent, and thereafter the dispensing of a protein solution to the dried media to initiate crystallization from the solid–liquid interface. Not only quick and easy to perform, the method also allows for a less concentrated protein solution for setting up crystallization trials.

The 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC) catalyses the decarboxylation of 3-keto-L-gulonate 6-phosphate to L-xylulose in the presence of magnesium ions. The enzyme is involved in L-ascorbate metabolism and plays an essential role in the pathway of glucuronate interconversion. Crystal structures of Streptococcus mutans KGPDC were determined in the absence and presence of the product analog D-ribulose 5-phosphate. We have observed an 8 A alphaB-helix movement and other structural rearrangements around the active site between the apo-structures and product analog bound structure. These drastic conformational changes upon ligand binding are the first observation of this kind for the KGPDC family. The flexibilities of both the alpha-helix lid and the side chains of Arg144 and Arg197 are associated with substrate binding and product releasing. The open-closed conformational changes of the active site, through the movements of the alpha-helix lid and the arginine residues are important for substrate binding and catalysis.

The punA gene of the cariogenic pathogen Streptococcus mutans encodes purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, catalyzing the phosphorolysis of purine nucleosides to generate purine bases and alpha-ribose 1-phosphate. In the present work, the PNP protein was expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique; the crystals diffracted to 1.6 A resolution at best. The crystals belonged to space group H3, with unit-cell parameters a = b = 113.0, c = 60.1 A.

As an oral bacterial pathogen, Streptococcus mutans has been known as the aetiologic agent of human dental caries. Among a total of 1960 identified proteins within the genome of this organism, there are about 500 without any known functions. One of these proteins, SMU.440, has very few homologs in the current protein databases and it does not fall into any protein functional families. Phylogenetic studies showed that SMU.440 is related to a particular ecological niche and conserved specifically in some oral pathogens, due to lateral gene transfer. The co-occurrence of a MarR protein within the same operon among these oral pathogens suggests that SMU.440 may be associated with antibiotic resistance. The structure determination of SMU.440 revealed that it shares the same fold and a similar pocket as polyketide cyclases, which indicated that it is very likely to bind some polyketide-like molecules. From the interlinking structural and bioinformatics studies, we have concluded that SMU.440 could be involved in polyketide-like antibiotic resistance, providing a better understanding of this hypothetical protein. Besides, the combination of multiple methods in this study can be used as a general approach for functional studies of a protein with unknown function.

Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for ab initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5 A resolution using Cr Kalpha radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family.

A wide range of bacteria secrete proteases into their extracellular environment for various purposes, such as degrading extracellular proteins for facilitating nutrient transport or effecting bacterial virulence and toxicity.1 A bacterial secreted protease is normally composed of a secretion signal peptide, a propeptide which will be cleaved upon the protease activation, and a mature secreted protease. The mature protease is the functional enzyme and can be isolated and characterized from the extracellular medium. This article concerns an endoproteinase secreted by an alkaliphilic and moderately halophilic microbe belonging to the Nesterenkonia abyssinica family (originally named as Nesterenkonia sp. AL20). This protease, designated as NAALP (Nesterenkonia abyssinica alkaline protease), was isolated from an alkaline soda lake in the East African rift valley.2, 3 The bacterium AL20 grows well with chicken feather as nutrient source, and the NAALP has shown good activity towards casein and hemoglobin as substrates in vitro, with sequence preference in the order of Tyr > Phe > Leu at the P1 site.4 Although activity profiles of the NAALP suggested that the enzyme to be a subtilisin-like protease, its activity and stability were calcium independent. The NAALP is optimally active at pH 10, 1.0M NaCl, and 70°C and shows good stability at 50°C in the presence of EDTA and detergents.5 With the information of hundreds of bacterial genomes available in the postgenomic era, thousands of novel proteins, annotated as open reading frames (ORFs), have been identified without biochemical characterization. Sequence searches using the NAALP as probe have revealed dozens of homologues in the sequenced bacterial genomes, the overwhelming majority of which are uncharacterized putative proteins, thus the NAALP has defined a novel protein family (defined by sequence identity over 30% to ensure the same structural fold) of bacterial secreted proteases. In this report, using high resolution crystal structure determination, we have unambiguously characterized the NAALP and its sequence related family as a trypsin-like serine protease. The sequence of the mature enzyme of NAALP was used to search the European Bioinformatics Institute (EBI) UniProt Knowledgebase at the website: http://www.ebi.ac.uk/fasta33/. Representative homologous sequences from FASTA searched results were selected and aligned with the program CLUSTALX.6 The protein preparation, crystallization, and diffraction data collection have been described.7 There are two molecules per asymmetric unit, and a two-fold noncrystallographic symmetry (NCS) was revealed by self-rotational analysis. The crystal structure determination was carried out by molecular replacement (MR) method using the program MolRep in the CCP4 package.8 The crystal structure of a glutamyl endopeptidase (with a sequence identity of 22%, the closest homolog of NAALP could be found in the PDB database) from Bacillus intermedius (PDB ID: 1P3E) was used as the searching model.9 Based on the sequence alignment, several different constructs of 1P3E were prepared for MR. For each construct, poly-alanine, poly-serine, and partial mutation models were tested with MolRep by exactly the same protocol. Self-rotation results were input for MR search in the range of 20–3 Å. The final solution was determined with the poly-serine model of residues 20–215 from 1P3E_chain A. All the top 30 rotation peaks were used for translation searches. The solution for one molecule was solved from the first rotation peak, which was confirmed by a quite sharp translation peak (TF/sig value 6.7, whereas the following peaks were around 3.7). The position of the second molecule was searched by fixing the first one, and the resulted dimer was subjected to refinement. After rigid body refinement by MolRep, the program ARP/wARP was used for further refinement and automatic model tracing.10 Refinement of the high resolution (1.39 Å) structure was carried out with the program Refmac5 combined with manually rebuilding of the model by the graphical program COOT.11, 12 Stereochemistry qualities of the model were evaluated and checked by PROCHECK.13 The data collection and structure refinement statistics were listed in Table I. Structure factors and the coordinates have been deposited in the PDB (PDB ID: 3CP7). Among about 100 returned search results, more than 95% sequences were previously uncharacterized putative proteins or ORFs, we have used the criteria of 30% sequence identity to define the NAALP-like family, about 30 sequences were selected and all of them were annotated as putative proteins without any biochemical characterization except for NAALP. Hence, this NAALP-like family belongs to a novel family of bacterial secreted serine proteases. Thirteen sequences (including NAALP itself) were selected as representatives of this NAALP-like family to do a structure-based multiple sequence alignment by CLUSTALX as shown in Figure. 1(A). The selected protein sequences are from the following bacterium organism sources: A0JTW5_ARTS2: Arthrobacter sp.; A1R423_ARTAT: Arthrobacter aurescens; A8L163_9ACTO: Frankia sp. EAN1pec; Q2J634_FRASC: Frankia sp.; Q47RY4_THEFY: Thermobifida fusca; Q8EM66_OCEIH: Oceanobacillus iheyensis; Q6AG07_LEIXX: Leifsonia xyli; Q2MG24_ MICEC: Micromonospora echinospora; A8M1I5_SALAI: Salinispora arenicola; A0LN25_SYNFM: Syntrophobacter fumaroxidans; A6W7 × 5_KINRD: Kineococcus radiotolerans; A8CXG6_9CHLR: Dehalococcoides sp. Figure 1(A) has clearly shown that all the structural elements, and functional important residues including the active site triad (S169, H41 and D91 as numbered in NAALPs) labeled by filled squares, the oxyanion hole labeled by filled rings, and the two pairs of intramolecular disulfide bridges, C23-C42, C144-C162 are very well conserved in the NAALP-like family. (A) Structure-based multiple sequence alignment of the NAALP family with representative members selected, see text for detailed names of each microorganism. The active site triad (S169, H41, and D91 as numbered in NAALPs) is labeled by filled squares, the oxyanion hole labeled by filled rings, and the two pairs of intramolecular disulfide bridges, C23-C42, C144-C162 were labeled by green numbers. The figure was produced by ESPript 2.2 (http://endscript.ibcp.fr/ESPript/ESPript/). (B) Topology diagram of the NAALP structure colored according to the secondary structure elements and different domains, with all helices red, and β strands green (domain I) and yellow (domain II). (C) Cα trace of the NAALP dimer showing Mol A in yellow and Mol B in green, helices α1 and α3 forming the dimer interfaces predominantly. (D) Detailed Cα trace of Mol A of Figure 1D with the secondary structure elements numbered, the active site triad labeled as S169, H41, and D91 and the two pairs of intramolecular disulfide bridges labeled as C23-C42, C144-C162, with C atoms in yellow, N atoms in blue, O atoms in red, and S atoms in green. (E) 2Fo-Fc density maps of the active site of molecule A at 1.0 σ. The active triad is labeled as His41, Asp91, and Ser169, with C atoms in green, N atoms in blue, O atoms in red, and density map in light blue. The oxyanion hole formed by the main-chain NH groups of Gly167 and Ser169. A formic acid molecule is in the active sites, with C atom in yellow and O atoms in red. The final model of NAALP is refined to 1.39 Å with R-factor and freeR-factor of 17.8% and 19.9%, respectively. Refinement statistics and model quality of NAALP are listed in Table I. The overall structure of NAALP contains two molecules in one asymmetry unit [Fig. 1(B), labeled as Mol A and Mol B], each molecule is very similar in three-dimensional structure and adopts a typical trypsin-like fold, consisting of two lobes, each formed by a six-stranded β-barrel. In addition to its beta protein features, NAALP also contains two short alpha helices, a longer α helix, α3, and three turns at the C-terminus [Fig. 1(B,C)]. In the crystal lattice, two molecules of NAALP pack together to form a crystallographic dimer with an interacting surface of about 880 Å2 which is in the range of a weak protein dimer (the interacting surface area of NAALP dimer was calculated at the website: http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver).14 The dimer interfaces are formed mainly by the α1 and α3 helices from both monomers packing against each other with predominantly H-bonds, salt bridges, and hydrophobic interactions [Fig. 1(B)]. In the previous studies, because the full-length sequence was not available, NAALP was identified as a subtilisin-like family of serine protease mainly due to biochemical features.2, 4 With further bioinformatics annotations after the structure was available, the NAALP has been assigned as trypsin-like from the CATH Protein Structure Classification database (http://www.cathdb.info) and Pfam Protein Families database (http://pfam.sanger.ac.uk). Furthermore, our structural results have undoubtedly shown that the NAALP is very similar to the trypsin in three-dimensional structure and all the functional elements for a trypsin-like serine protease are completely conserved, therefore the NAALP-like family has a trypsin-like structure and function. The active sites in both molecules of NAALP are very similar and readily identified as in the active form with the intact catalytic triad and oxyanion hole shown, labeled as S169, H41, D91 depicted in Figure 1(D,E). A formic acid molecule (existing in 2.9M sodium formate in the crystallization buffer) has been observed in both active sites, with an oxygen atom positioned in the oxyanion hole formed by the main-chain NH groups of Gly167 and Ser169, somewhat resembling the new carboxy terminus of a cleaved substrate.

Asx Te100 -x glasses with x ≤ 40 show single stage crystallization and those with x ≥ 40 exhibit a double stage crystallization and at x = 40, this is associated with "rigidity percolation" and "chemical stoichiometric ordering".In the present study the effect of pressure on the thermal crystallization of Asx Te100 -x, Asx Te100 -x -y Se y glasses has been investigated by differential thermal analyzer at high pressure (HP-DTA).For As = 40 and 50 system, in Asx Te100 -x and Asx Te100 -x -y Sey, the first exothermic peaks are converted to endothermic under pressure and this is considered as rigidity percolation.The second exothermic peak do not converted to endothermic or no structural transformation takes place.This is considered as electron localization to delocalization.In As = 30, 40 and 50 system, as the Se content increases, the volume decreases from the initial value and the shifting of the temperature of the peaks reduces than the basic system because of less structural transformation.Thus it is concluded that the second peak is generated because of the electron localization.

Sessions remains poorly understood.The enzyme used in this study is papain, a member of the C1 family and the archetypal CP.Papain is obtained from papaya fruits; its utility in tenderizing meat has been known for thousands of years.Papain finds immunological utility in the cleavage of immunoglobulins into Fc and FAB fragments, and medical use in the treatment of stings and chronic wounds.Earlier work reported the crystallization of papain from ethanol/methanol solutions, revealing that papain comprises 2 major structural domains.Papain activation is believed to depend on the formation of a thiolate-imidazolium pair between residues Cys25 and His159 at the cleft between domains.In this study we have obtained crystals from a new aqueous condition containing PEG, buffer, and sodium thiosulfate.In this condition, papain crystallizes in a low-solventcontent unit cell.A 1.60 Å X-ray data set was collected at 300 K in 4 h on a copper-source diffractometer.Results show that a thiosulfate moiety is bound to the active site cysteine, Cys25.Efforts to optimize crystal size for ultra-high resolution X-ray diffraction and neutron diffraction data collection are ongoing.By locating the hydrogen atoms at the active site, we hope to determine the protonation state of His159 and obtain a clearer picture of papain activation and substrate hydrolysis.

As part of a structural genomics platform in a university laboratory, a low-cost in-house-developed automated imaging system for SBS microplate experiments has been designed and constructed. The imaging system can scan a microplate in 2-6 min for a 96-well plate depending on the plate layout and scanning options. A web-based crystallization database system has been developed, enabling users to follow their crystallization experiments from a web browser. As the system has been designed and built by students and crystallographers using commercially available parts, this report is aimed to serve as a do-it-yourself example for laboratory robotics.

A large- scale, high- efficiency and low- cost platform based on a Beckman Coulter Biomek FX and custom- made automation systems for structural genomics has been set up at Peking University, Beijing, People's Republic of China. This platform has the capacity to process up to 2000 genes per year for structural and functional analyses. Bacillus subtilis, a model organism for Gram- positive bacteria, and Streptococcus mutans, a major pathogen of dental caries, were selected as the main targets. To date, more than 470 B. subtilis and 1200 S. mutans proteins and hundreds of proteins from other sources, including human liver proteins, have been selected as targets for this platform. The selected genes are mainly related to important metabolism pathways and/ or have potential relevance for drug design. To date, 40 independent structures have been determined; of these 11 are in the category of novel structures by the criterion of having less than 30% sequence identity to known structures. More than 13 structures were determined by SAD/ MAD phasing. The macromolecular crystallography beamline at the Beijing Synchrotron Radiation Facility and modern phasing programs have been crucial components of the operation of the platform. The idea and practice of the genomic approach have been successfully adopted in a moderately funded structural biology program and it is believed this adaptation will greatly improve the production of protein structures. The goal is to be able to solve a protein structure of moderate difficulty at a cost about US $ 10 000.

Imidazolonepropionase (EC 3.5.2.7) catalyzes the third step in the universal histidine degradation pathway, hydrolyzing the carbon-nitrogen bonds in 4-imidazolone-5-propionic acid to yield N-formimino-L-glutamic acid. Here we report the crystal structures of the Bacillus subtilis imidazolonepropionase and its complex at 2.0-A resolution with substrate analog imidazole-4-acetic acid sodium (I4AA). The structure of the native enzyme contains two domains, a TIM (triose-phosphate isomerase) barrel domain with two insertions and a small beta-sandwich domain. The TIM barrel domain is quite similar to the members of the alpha/beta barrel metallo-dependent hydrolase superfamily, especially to Escherichia coli cytosine deaminase. A metal ion was found in the central cavity of the TIM barrel and was tightly coordinated to residues His-80, His-82, His-249, Asp-324, and a water molecule. X-ray fluorescence scan analysis confirmed that the bound metal ion was a zinc ion. An acetate ion, 6 A away from the zinc ion, was also found in the potential active site. In the complex structure with I4AA, a substrate analog, I4AA replaced the acetate ion and contacted with Arg-89, Try-102, Tyr-152, His-185, and Glu-252, further defining and confirming the active site. The detailed structural studies allowed us to propose a zinc-activated nucleophilic attack mechanism for the hydrolysis reaction catalyzed by the enzyme.