A response to the article by Rupp (2012), Acta Cryst. F68, 366–376.

The authors of J. Immunol. 184, 725–735 respond to the article by Rupp (2012), Acta Cryst. F68, 366–376.

Editorial.

Development of an ontology for the description of crystallization experiments and results is proposed.

When crystallization screening is conducted many outcomes are observed but typically the only trial recorded in the literature is the condition that yielded the crystal(s) used for subsequent diffraction studies. The initial hit that was optimized and the results of all the other trials are lost. These missing results contain information that would be useful for an improved general understanding of crystallization. This paper provides a report of a crystallization data exchange (XDX) workshop organized by several international large-scale crystallization screening laboratories to discuss how this information may be captured and utilized. A group that administers a significant fraction of the world’s crystallization screening results was convened, together with chemical and structural data informaticians and computational scientists who specialize in creating and analysing large disparate data sets. The development of a crystallization ontology for the crystallization community was proposed. This paper (by the attendees of the workshop) provides the thoughts and rationale leading to this conclusion. This is brought to the attention of the wider audience of crystallographers so that they are aware of these early efforts and can contribute to the process going forward.

The structure of L. donovani pteridine reductase has been targeted to assist in a program of structure-based inhibitor research. Crystals that diffracted to 2.5 Å resolution were obtained and the structure has been solved. Unfortunately, the active site is disordered and this crystal form is unsuitable for use in characterizing enzyme–ligand interactions.

Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes.

MTH909 is the Methanothermobacter thermautotrophicus ortholog of Saccharomyces cerevisiae TAN1, which is required for N4-acetylcytidine formation in tRNA. The protein consists of an N-terminal near ferredoxin-like domain and a C-terminal THUMP domain. Unlike most other proteins containing the THUMP domain, TAN1 lacks any catalytic domains and has been proposed to form a complex with a catalytic protein capable of making base modifications. MTH909 has been cloned, over-expressed and purified. The molecule exists as a monomer in solution. X-ray data from a native crystal, belonging to the space group P6122 (P6522) with the unit cell dimensions of a = 69.9 Å and c = 408.5 Å, have been collected to 2.85 Å resolution.

Synopsis

MTH909, the Methanothermobacter thermautotrophicus ortholog of Saccharomyces cerevisiae TAN1, has been over-expressed, purified and crystallized. X-ray data from a crystal belonging to the space group P6122 (P6522) have been collected to 2.85 Å resolution.

The carbohydrate-binding region of GspB from S. gordonii strain M99 was crystallized in space group P212121 and data were collected to 1.3 Å resolution.

The carbohydrate-binding region of the bacterial adhesin GspB from Strepto­coccus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 Å for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 Å for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall R merge value of 0.04 and an R merge value of 0.33 in the highest resolution shell.

The carbohydrate binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts, and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, these had distinct unit cell dimensions of a=33.3 Å, b=86.6 Å, c=117.9 Å for crystal form one and a=34.6 Å, b=98.3 Å, c=99.0 Å for crystal form two. Additive screening improved the crystals grown in both conditions such that diffraction extended beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall Rsym value of 0.04 and an Rsym value of 0.33 in the highest resolution shell.

The major outer membrane protein PorB from N. meningitidis was crystallized in three crystal forms; the best X-ray diffraction data were collected to 2.3 Å resolution.

The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P63. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P63 crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P63 crystal form had the best diffraction quality, yielding data extending to 2.3 Å resolution.

The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P63. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit while both the R32 and P63 crystal forms contain one PorB monomer in the asymmetric unit. Of the three, the P63 crystal form had the best diffraction quality, yielding data extending to 2.3 Å resolution.

Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in Mycobacterium spp. Here, the crystallization and preliminary crystallographic analysis of GpgS from M. tuberculosis and of its complex with UDP are reported.

Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty-acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active Enzymes Database; see http://www.cazy.org). Here, the purification, crystallization and preliminary crystallographic analysis are reported of GpgS from Mycobacterium tuberculosis and of its complex with UDP. GpgS crystals belonged to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c = 127.64 Å, and diffracted to 2.6 Å resolution. GpgS–UDP complex crystals belonged to space group I4, with unit-cell parameters a = 98.32, b = 98.32, c = 127.96 Å, and diffracted to 3.0 Å resolution.

Three data sets have been collected on endothiapepsin complexed with the gem-diol inhibitor PD-135,040: a high-resolution synchrotron X-ray data set, a room-temperature X-ray data set and a neutron diffraction data set. Until recently, it has been impossible to grow large protein crystals of endothiapepsin with any gem-diol inhibitor that are suitable for neutron diffraction.

Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme–inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.

Synopsis Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in Mycobacterium spp. Here we report the crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP at 2.6 Å and 3.0 Å resolution, respectively.

Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active enZymes data base; see www.cazy.org). Here we report the purification, crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP. GpgS crystals belong to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c= 127.64 Å, and diffract to 2.6 Å resolution. GpgS-UDP complex crystals belong to space group I4 with unit-cell parameters a= 98.32, b= 98.32, c= 127.96 Å, and diffract to 3.0 Å resolution.

The NKX2.5 homeodomain has been crystallized in complex with DNA. Diffraction data were collected to 1.7 Å resolution.

As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the −242 promoter region of atrial natriuretic factor. Crystals of the homeodomain–DNA complex diffracted X-rays to 1.7 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 71.5, c = 94.3 Å. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.

The X-ray crystal structure of a 6-pyruvoyltetrahydropterin synthase homolog of unknown function has been determined at 1.5 Å resolution. The protein retains residues required for pterin binding, but nearly all catalytic residues are missing.

The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate.

The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate.

Histamine dehydrogenase from Nocardioides simplex has been expressed, purified and crystallized with full incorporation of 6-S-cysteinyl-FMN. Diffraction data have been collected to 2.7 Å resolution; the crystals belonged to the orthorhombic space group P212121.

Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe–4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 Å resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 Å.

Prolyl-4-hydroxylase from B. anthracis has been cloned, expressed and crystallized. A complete MAD data set has been collected to 1.4 Å resolution.

Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 Å resolution.

Collagen prolyl-4-hydroxylases (C-P4H) catalyze the hydroxylation of specific proline residues in procollagen, an essential step in collagen biosynthesis. We have recently characterized a new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human. The structure of anthrax-P4H could provide important insights into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in E. coli has been collected to 1.4 Å resolution.

Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and ammonium. HADH is functionally related to trimethylamine dehydrogenase (TMADH) but HADH has strict substrate specificity towards histamine. HADH is a homodimer with each 76 kDa subunit containing two redox cofactors, a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, we seek to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 Å resolution at the SSRL synchrotron with 99.7% completeness. The crystals belong to the orthorhombic space group P212121 with unit-cell parameters a = 101.14 Å, b = 107.03 Å, and c = 153.35 Å.

A 3.02 Å crystal structure of native GroEL from E. coli is presented.

GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 Å crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes that may occur in response to cognate potassium-ion binding by comparison to the previously determined wild-type GroEL structure (PDB code 1xck), in which potassium ions were observed in all 14 subunits. In general, the structure is similar to the previously determined wild-type GroEL crystal structure with some differences in regard to temperature-factor distribution.

In order to investigate the role of the protonation states of protein residues in O2 binding, large crystals of deoxy HbA (∼20 mm3) were grown in D2O under anaerobic conditions for neutron diffraction studies.

Human hemoglobin (HbA) is an intricate system that has evolved to efficiently transport oxygen molecules (O2) from lung to tissue. Its quaternary structure can fluctuate between two conformations, T (tense or deoxy) and R (relaxed or oxy), which have low and high affinity for O2, respectively. The binding of O2 to the heme sites of HbA is regulated by protons and by inorganic anions. In order to investigate the role of the protonation states of protein residues in O2 binding, large crystals of deoxy HbA (∼20 mm3) were grown in D2O under anaerobic conditions for neutron diffraction studies. A time-of-flight neutron data set was collected to 1.8 Å resolution on the Protein Crystallography Station (PCS) at the spallation source run by Los Alamos Neutron Science Center (LANSCE). The HbA tetramer (64.6 kDa; 574 residues excluding the four heme groups) occupies the largest asymmetric unit (space group P21) from which a high-resolution neutron data set has been collected to date.

Human hemoglobin (HbA) is an intricate system that has evolved to efficiently transport oxygen molecules (O2) from lung to tissue. Its quaternary structure can fluctuate between two conformations, T (tense or deoxy) and R (relaxed or oxy), which have low and high affinity for O2, respectively. The binding of O2 to the heme sites of HbA is regulated by protons and by inorganic anions. In order to investigate the role of the protonation states of protein residues in O2 binding, large crystals of deoxy HbA (∼20 mm3) were grown in D2O under anaerobic conditions for neutron diffraction studies. A time-of-flight neutron data set was collected to 1.8Å resolution on the Protein Crystallography Station (PCS) at the spallation source run by Los Alamos Neutron Science Center (LANSCE). The HbA tetramer (64.6 kDa; 574 residues excluding the four heme groups) occupies the largest asymmetric unit (space group P21) from which a high-resolution neutron data set has been collected to date.

Diisopropyl fluorophosphatase (DFPase) effectively hydrolyzes a number of organophosphorus nerve agents, including sarin, cyclohexylsarin, soman and tabun. Neutron diffraction data have been collected from DFPase crystals to 2.2 Å resolution in an effort to gain further insight into the mechanism of this enzyme.

The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 Å resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the −242 promoter region of atrial natriuretic factor. Crystals of the homeodomain–DNA complex diffracted X-rays to 1.7 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 71.5, c = 94.3 Å. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.

Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme—inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.