Crystallographic education in the 21st century.

There are many methods that can be used to incorporate concepts of crystallography into the learning experiences of students, whether they are in elementary school, at university or part of the public at large. It is not always critical that those who teach crystallography have immediate access to diffraction equipment to be able to introduce the concepts of symmetry, packing or molecular structure in an age- and audience-appropriate manner. Crystallography can be used as a tool for teaching general chemistry concepts as well as general research techniques without ever having a student determine a crystal structure. Thus, methods for younger students to perform crystal growth experiments of simple inorganic salts, organic compounds and even metals are presented. For settings where crystallographic instrumentation is accessible (proximally or remotely), students can be involved in all steps of the process, from crystal growth, to data collection, through structure solution and refinement, to final publication. Several approaches based on the presentations in the MS92 Microsymposium at the IUCr 23rd Congress and General Assembly are reported. The topics cover methods for introducing crystallography to undergraduate students as part of a core chemistry curriculum; a successful short-course workshop intended to bootstrap researchers who rely on crystallography for their work; and efforts to bring crystallography to secondary school children and non-science majors. In addition to these workshops, demonstrations and long-format courses, open-format crystallographic databases and three-dimensional printed models as tools that can be used to excite target audiences and inspire them to pursue a deeper understanding of crystallography are described.

Structural Basis for Enhancement of Carbapenemase Activity in the OXA-51 Family of Class D ß-Lactamases.

Class D ß-lactamases of Acinetobacter baumannii are enzymes of the utmost clinical importance, producing resistance to last resort carbapenem antibiotics. Although the OXA-51-like enzymes constitute the largest family of class D ß-lactamases, they are poorly studied and their importance in conferring carbapenem resistance is controversial. We present the detailed microbiological, kinetic, and structural characterization of the eponymous OXA-51 ß-lactamase. Kinetic studies show that OXA-51 has low catalytic efficiency for carbapenems, primarily due to the low affinity of the enzyme for these substrates. Structural studies demonstrate that this low affinity results from the obstruction of the enzyme active site by the side chain of Trp222, which presents a transient steric barrier to an incoming carbapenem substrate. The Trp222Met substitution relieves this steric hindrance and elevates the affinity of the mutant enzyme for carbapenems by 10-fold, significantly increasing the levels of resistance to these antibiotics. The ability of OXA-51 to evolve into a robust carbapenemase as the result of a single amino acid substitution may, in the near future, elevate the ubiquitous enzymes of the OXA-51 family to the status of the most deleterious A. baumannii carbapenemases, with dire clinical consequences.

Synthesis, structures, spectroscopic and electrochemical properties of dinitrosyl iron complexes with bipyridine, terpyridine, and 1,10-phenathroline.

Three new dinitrosyl iron complexes LFe(NO)(2) (L = 2,2'-bipyridine (bipy) (1), 2,2',2''-terpyridine (terpy) (2) and 1,10-phenathroline (phen) (3)) were synthesized by the reaction of Fe(NO)(2)(CO)(2) with corresponding ligands in tetrahydrofuran. Complexes 1-3 were studied using IR, UV-vis, MS, NMR, and electrochemical techniques. Complexes 1 and 2 were also characterized using single crystal X-ray diffraction analysis. IR spectra of complexes 1-3 display two strong characteristic NO stretching frequencies (nu(NO)) in the region reflecting donor properties of the ligands. Cyclic voltammetry studies show two quasi-reversible one-electron reductions for all complexes. Electrochemical investigations using different concentrations show that an irreversible one-electron reduction at -1.85 V for complex 2 and -1.80 V for complex 3 are from solvated species. Single-crystal X-ray structural analysis reveals that complex 1 crystallizes in the triclinic P1 space group and the asymmetric unit consists of one Fe(NO)(2)(bipy) molecule with the two NO groups located on two sides of Fe(bipy) plane. Complex 2 crystallizes in monoclinic P21/n space group, and the asymmetric unit contains one Fe(NO)(2)(terpy) molecule, in which the NO groups are located on two sides of the plane consisted of Fe and two coordinated pyridyl rings, but almost parallel to the uncoordinated pyridyl ring. The crystal packings of both complexes 1 and 2 show intermolecular H-bonding and strong pi-pi stacking interactions.

Structure of GES-1 at atomic resolution: insights into the evolution of carbapenamase activity in the class A extended-spectrum beta-lactamases.

The structure of the class A extended-spectrum beta-lactamase GES-1 from Klebsiella pneumoniae has been determined to 1.1 A resolution. GES-1 has the characteristic active-site disulfide bond of the carbapenemase family of beta-lactamases and has a structure that is very similar to those of other known carbapenemases, including NMC-A, SME-1 and KPC-2. Most residues implicated in the catalytic mechanism of this class of enzyme are present in the GES-1 active site, including Ser70, which forms a covalent bond with the carbonyl C atom of the beta-lactam ring of the substrate during the formation of an acyl-enzyme intermediate, Glu166, which is implicated as both the acylation and deacylation base, and Lys73, which is also implicated as the acylation base. A water molecule crucial to catalysis is observed in an identical location as in other class A beta-lactamases, interacting with the side chains of Ser70 and Glu166. One important residue, Asn170, also normally a ligand for the hydrolytic water, is missing from the GES-1 active site. This residue is a glycine in GES-1 and the enzyme is unable to hydrolyze imipenem. This points to this residue as being critically important in the hydrolysis of this class of beta-lactam substrate. This is further supported by flexible-docking studies of imipenem with in silico-generated Gly170Asn and Gly170Ser mutant GES-1 enzymes designed to mimic the active sites of imipenem-hydrolyzing point mutants GES-2 and GES-5.

Dialkyl phenyl phosphates as novel selective inhibitors of butyrylcholinesterase.

A series of dialkyl phenyl phosphates (DAPPs) were synthesized and evaluated in silico and in vitro for inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Among the compounds examined, several DAPPs were shown to be potent inhibitors of butyrylcholinesterase, while having little activity against acetylcholinesterase. The most potent and selective inhibitors were di-n-butyl phenyl phosphate (K(i)=43 microM), di-n-pentyl phenyl phosphate (K(i)=6 microM), and di-cyclohexyl phenyl phosphate (K(i)=7 microM), the first which was shown to be a competitive inhibitor while the latter two being partial competitive inhibitors. Flexible docking simulations suggested that relative binding affinities generally increased as a function of alkyl chain length, while the strength and nature of inhibitory activity depended on whether the compound bound deeply or midway in the active site gorge, or in the proposed peripheral site.

Molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality.

BACKGROUND: Human rhinoviruses (HRVs) are the most frequent cause of acute upper respiratory tract infection, however, they are also known to replicate in the lower respiratory tract and associate with more severe respiratory illnesses. An outbreak of HRV occurred in a long-term facility in Santa Cruz, California with unusually high morbidity and mortality. OBJECTIVES: To identify viral characteristics associated with this unique outbreak, genetic relationships between these clinical isolates (SCRVs) and prototype strains of rhinovirus were investigated. STUDY DESIGN: Sequence homology and phylogenetic analyses of the SCRV VP4/VP2 region were performed in conjunction with all HRV prototypes. Due to the importance of the 5'noncoding region (NCR) and the structural genes to viral replication and host immune responses, respectively, we focused on a segment of the HRV genome which includes these regions. Molecular models of SCRV were also assessed. RESULTS: SCRV showed closest similarity to HRV82 with some divergence from the prototype. Amino acid differences were concentrated within predicted neutralization epitopes within VP2, VP3 and VP1. CONCLUSION: Sequence analyses and differences in cell culture growth characteristics suggest that this virus is a variant of HRV which has distinctive properties from its respective prototype strain.

Structure of pyrR (Rv1379) from Mycobacterium tuberculosis: a persistence gene and protein drug target.

The Mycobacterium tuberculosis pyrR gene (Rv1379) encodes a protein that regulates the expression of pyrimidine-nucleotide biosynthesis (pyr) genes in a UMP-dependent manner. Because pyrimidine biosynthesis is an essential step in the progression of TB, the gene product pyrR is an attractive antitubercular drug target. The 1.9 A native structure of Mtb pyrR determined by the TB Structural Genomics Consortium facilities in trigonal space group P3(1)21 is reported, with unit-cell parameters a = 66.64, c = 154.72 A at 120 K and two molecules in the asymmetric unit. The three-dimensional structure and residual uracil phosphoribosyltransferase activity point to a common PRTase ancestor for pyrR. However, while PRPP- and UMP-binding sites have been retained in Mtb pyrR, a distinct dimer interaction among subunits creates a deep positively charged cleft capable of binding pyr mRNA. In silico screening of pyrimidine-nucleoside analogs has revealed a number of potential lead compounds that, if bound to Mtb pyrR, could facilitate transcriptional attenuation, particularly cyclopentenyl nucleosides.

A cyclic tetra-nuclear dinitrosyl iron complex [Fe(NO)2(imidazolate)]4: synthesis, structure and stability.

A novel cyclic tetra-nuclear dinitrosyl iron complex [Fe(NO)2(Im-H)]4 was isolated and characterized by X-ray crystallography, and in donor solvents this fragments into 17 e- monomeric units that give EPR spectra analogous to the g= 2.03 species seen in mammalian biology.

Laboratory scale structural genomics.

At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crystallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and challenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and protocols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment.

Mycobacterium tuberculosis RmlC epimerase (Rv3465): a promising drug-target structure in the rhamnose pathway.

The Mycobacterium tuberculosis rmlC gene encodes dTDP-4-keto-6-deoxyglucose epimerase, the third enzyme in the M. tuberculosis dTDP-L-rhamnose pathway which is essential for mycobacterial cell-wall synthesis. Because it is structurally unique, highly substrate-specific and does not require a cofactor, RmlC is considered to be the most promising drug target in the pathway, and the M. tuberculosis rmlC gene was selected in the initial round of TB Structural Genomics Consortium targets for structure determination. The 1.7 A native structure determined by the consortium facilities is reported and implications for in silico screening of ligands for structure-guided drug design are discussed.

Protein isoelectric point as a predictor for increased crystallization screening efficiency.

MOTIVATION: Increased efficiency in initial crystallization screening reduces cost and material requirements in structural genomics. Because pH is one of the few consistently reported parameters in the Protein Data Bank (PDB), the isoelectric point (pI) of a protein has been explored as a useful indirect predictor for the optimal choice of range and distribution of the pH sampling in crystallization trials. RESULTS: We have analyzed 9596 unique protein crystal forms from the August 2003 PDB and have found a significant relationship between the calculated pI of successfully crystallized proteins and the difference between pI and reported pH at which they were crystallized. These preferences provide strong prior information for the design of crystallization screening experiments with significantly increased efficiency and corresponding reduction in material requirements, leading to potential cost savings of millions of US$ for structural genomics projects involving high-throughput crystallographic structure determination. AVAILABILITY: A prototype example of a screen design and efficiency estimator program, CrysPred, is available at http://www-structure.llnl.gov/cryspred/

Effective electron-density map improvement and structure validation on a Linux multi-CPU web cluster: The TB Structural Genomics Consortium Bias Removal Web Service.

Anticipating a continuing increase in the number of structures solved by molecular replacement in high-throughput crystallography and drug-discovery programs, a user-friendly web service for automated molecular replacement, map improvement, bias removal and real-space correlation structure validation has been implemented. The service is based on an efficient bias-removal protocol, Shake&wARP, and implemented using EPMR and the CCP4 suite of programs, combined with various shell scripts and Fortran90 routines. The service returns improved maps, converted data files and real-space correlation and B-factor plots. User data are uploaded through a web interface and the CPU-intensive iteration cycles are executed on a low-cost Linux multi-CPU cluster using the Condor job-queuing package. Examples of map improvement at various resolutions are provided and include model completion and reconstruction of absent parts, sequence correction, and ligand validation in drug-target structures.

Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein-nucleic acid complex crystals.

Estimating the number of molecules in the crystallographic asymmetric unit is one of the first steps in a macromolecular structure determination. Based on a survey of 15641 crystallographic Protein Data Bank (PDB) entries the distribution of V(M), the crystal volume per unit of protein molecular weight, known as Matthews coefficient, has been reanalyzed. The range of values and frequencies has changed in the 30 years since Matthews first analysis of protein crystal solvent content. In the statistical analysis, complexes of proteins and nucleic acids have been treated as a separate group. In addition, the V(M) distribution for nucleic acid crystals has been examined for the first time. Observing that resolution is a significant discriminator of V(M), an improved estimator for the probabilities of the number of molecules in the crystallographic asymmetric unit has been implemented, using resolution as additional information.

Concanavalin A in a dimeric crystal form: revisiting structural accuracy and molecular flexibility.

A structure of native concanavalin A (ConA), a hardy perennial of structural biology, has been determined in a dimeric crystal form at a resolution of 1.56 A (space group C222(1); unit-cell parameters a = 118.70, b = 101.38, c = 111.97 A; two molecules in the asymmetric unit). The structure has been refined to an R(free) of 0.206 (R = 0.178) after iterative model building and phase-bias removal using Shake&wARP. Correspondence between calculated water-tyrosine interactions and experimentally observed structures near the saccharide-binding site suggests that the observed interactions between Tyr12 and water in various crystal forms are to be expected and are not unique to the presence of an active site. The present structure differs from previously reported atomic resolution structures of ConA in several regions and extends insight into the conformational flexibility of this molecule. Furthermore, this third, low-temperature, structure of ConA in a different crystal form, independently refined using powerful model-bias removal techniques, affords the opportunity to revisit assessment of accuracy and precision in high- or atomic resolution protein structures. It is illustrated that several precise structures of the same molecule can differ substantially in local detail and users of crystallographic models are reminded to consider the potential impact when interpreting structures. Suggestions on how to effectively represent ensembles of crystallographic models of a given molecule are provided.