ALBERT

Notes: In this paper I propose that spatially-informed and agency-centred treatments of prostitution as a practice can fruitfully highlight the moral and symbolic economies operating in the lives of women as they engage and disengage with prostitution in the tourist sex trade zones of Bangkok. By reexamining and re-contextualising the practices of prostitution in terms of interacting spatial/socio-cultural fields, we can gain more useful insights than heretofore obtained through a mono-dimensional political economy or gender-culture framework. We must accept that women sex workers are part of the geography of socio-economic transformation, responding to the centralisation and expansion of key sectors of the Thai economy. I conceptualise this broader geography as involving “oscillation” between urban and rural “places” (which represent key “fields” of value), and at the level of the city (and the tourist leisure infrastructure) as “movement” between different work sites and “arenas” of identity formation. Within this complex of circulation and engagement, women strategise to maximise what may be described as survival and status resources (conversion of income into cultural capital) aiming towards recovery of meaningful self-identity.

Notes: Recent research on how tourism has affected indigenous culture has steered away from the idea of “impact” and the disappearance of traditional culture to emphasising how local culture is created and negotiated within the context of tourism. This paper uses the latter approach, focusing on the way tourism is used in Western Flores to “authenticate” culture. The argument is that interaction with foreigners has opened up new ideas about what is “authentic” and “legitimate”, and therefore has created new spaces for contestation and negotiation. The development of tourism in Manggarai, the largest regency on Flores, has stimulated a number of villagers to rebuild traditional style houses as an attempt to attract foreign visitors. A few of these cases are examined here to show how the status of these houses is negotiated and contested within the context of a number of different ideas about authenticity.

Notes: In Malaysia, state promotion of tourism as a main component of service sector expansion and foreign exchange earnings has compelled government at all levels to facilitate leisure industry development. Even in the relatively small state of Melaka, where its historic landscapes constitute significant heritage tourism resources, development projects for theme parks and golf resorts have proliferated. This analysis examines Malaysian tourism development through an evaluation of the diverse tourism sectors in Melaka town and state—heritage, theme park, golf resort, and ecotourism—to demonstrate how the developmental contexts of national and international economic restructuring, combined with regional tourism demand for activity-oriented leisure, have rationalised large-scale, high-cost, environmentally-sensitive real estate development projects. In the process, authentic natural and historic landscapes have been rapidly forsaken for ersatz “leisurescapes” where both domestic and foreign tourists consume representations of diverse symbolic worlds.

Notes: Ecotourism, or tourism that promotes conservation, does not occur in isolation, but as part of an interdependent system. Based on a case study at Doi Inthanon National Park, Thailand, this study outlines a conceptual framework for such a system, placing biodiversity conservation in the context of other major tourism components, including tourists, ecotourism infrastructure, protected areas and local people. In an interactive way, each component affects the conservation of biodiversity. Based on a tourist survey and participant observation, we examine the interrelationships among the key tourism components and potential changes over time. Several environmental, social, economic and educational effects illustrate the complex nature of the ecotourism-conservation relationship. Recommendations to increase conservation benefits from ecotourism are provided.

Notes: Book reviewed in this article:Tourism and Sustainability: New Tourism in the Third World. Martin Mowforth and Ian MuntTourism and Politics: Policy, Power and Place. Colin Michael HallTourism and Economic Development in Asia and Australasia. Frank M. Go and Carson L. Jenkins (eds.), Tourism, Leisure, and Recreation SeriesIsland Tourism: Trends and Prospects. Douglas G. Lockhart and David Drakakis-Smith (eds.)Gender, Work and Tourism. M. Thea Sinclair (ed.)Place Matters: Gendered Geography in Victorian Women's Travel Books about Southeast Asia. Susan Morgan

Notes: This paper addresses the implications of the Asian economic crisis for tourism. The nature of the crisis is discussed briefly and its occurrence at a time of previously rapid regional growth in tourism is pointed out. It is argued that effective demand diminished at a time when supply was increasing. In the short term, reductions in regional travel and spending have been widespread and not confined to Southeast Asia. In the longer term, reduced real prices will make Asian destinations highly competitive in long-haul markets provided that the quality of products is maintained. The crisis emphasises the need to examine tourism planning initiatives, to acknowledge that the tourism product consists of more than the provision of quality accommodation, and to give greater attention to product differentiation.

Notes: This paper seeks to articulate recent debates on globalisation and critical security with the process and local impact of international tourism development in Thailand and Myanmar (Burma). It argues that, although tourism is an important manifestation of the globalisation phenomenon, its impact on host societies is very much dependent upon the influence of local institutions and actors. Hence, the enquiry will focus principally upon the “global-local interface”. The impact of tourism is viewed within the “new security” framework which involves broadening the notion of security beyond its traditional focus on military issues and inter-state relations into such realms as economic, environmental and societal security with a strong emphasis on sub-national situations. Using empirical examples of forced labour and human rights in Myanmar, the HIV/AIDS crisis and societal insecurity in Thailand and Myanmar, and the environmental impact of golf tourism in Thailand and elsewhere, the paper attempts to show how the perceived macro-level benefits of active tourism promotion are frequently traded off against significant, and occasionally severe, negative local security impacts. How such a situation occurs will be the principal focus of this investigation.

Notes: There are several good reasons why motorists should be charged for using roads, and it is important to understand these and to distinguish between them.Alternatives, however attractive, fail to stand up to close scrutiny. The change of government attitude showed in the recent White Paper is to be welcomed, but of the three specific proposals, two are questionable. Road pricing should not be used as a major welfare tool.

Notes: The recent White Paper indicates the start of serious attention to the use of road-use pricing, while the hard edge of the argument is to be found in the Treasury's Comprehensive Spending Review. What has yet to be examined is the problem of equipping the nation's vehicles with the necessary devices, effective for the purpose over the whole country. This could be done by replacing the paper ‘tax disc’ with a standardised electronic tag - carrying stored credit-units - which could provide for road-use payments as and where they come to be introduced.

Notes: This paper considers whether the current low inflation rates in the UK are due to‘the death of inflation’(because of structural changes to the economy, as claimed in a recent book) or should instead be interpreted as part of a business cycle which is largely monetary in origin.

Notes: Previously it was reported that regional variations in well-being (poverty, per capita income, and family income) among Appalachian counties did not originate from regional variations in urbanization, but from regional differences in well-being among nonmetropolitan counties. It was argued that southern Appalachian counties had higher levels of well-being at the end of the 1980s because nonmetropolian counties in southern Appalachia experienced greater economic growth during the 1980s than did nonmetropolitan counties in other Appalachian regions. In this paper these data are reanalyzed to test to what extent the original findings are affected by the presence (and failure to control) spatial autocorrelation. Using a spatial lag model it is shown that correcting for spatial autocorrelation statistically altered the original results. However, substantively, the conclusions from the original analysis did not change: regional differences in county well-being in Appalachia are largely the product of regional differences among nonmetropolitan counties, even after correcting the model for spatial autocorrelation.

Notes: Numerous reports have been published in the literature which describe the crystallization of macromolecules by a variety of crystallization methods, including the vapour-diffusion and microbatch techniques. This topical review compares the results of examples of proteins which were crystallized by both vapour-diffusion and microbatch methods. The inherent features of the vapour-diffusion and microbatch methods are discussed and some specific conditions where one method appears more favourable than the other are reported. Guidelines for the conversion of crystallization conditions from vapour diffusion to microbatch (and vice versa) are also presented.

Notes: The effects of a temperature increase on monoclinic and tetragonal lysozyme single crystals were investigated by polarizing microscopy, X-ray diffraction and laser Raman spectroscopy. To prevent dissolution, the mother liquor was removed, and the crystals were covered by the oil poly-(chlorotrifluoroethylene). Upon heating, their macroscopic shape was stable beyond 453 K but a change (or loss) of birefringence was observed around 352 and 367 K for the tetragonal and monoclinic crystal forms, respectively, which is associated with tighter packing and higher crystal forces in monoclinic lysozyme. Raman spectral changes in the amide I and amide III regions indicated denaturation of the protein within the crystalline environment at temperature where birefringence changes, and differences in the S—S band suggest that in monoclinic lysozyme, denaturation is accompanied with disruption of some S—S bonds. Comparison with thermal denaturation and gel formation (\beta-aggregation) of lysozyme in solution indicates that intermolecular interactions are mainly involved in the stabilization of the denatured lysozyme crystals. The behavior of ribonuclease A is very different. This protein unfolds and refolds reversibly in solution and its crystals melt at the unfolding temperature at 333 K, i.e. loss of structure induces breakdown of crystal lattice and macroscopic shape. Although the crystal lattice of proteins is stabilized by only few intermolecular contacts, its breakdown with increasing temperature is primarily a result of thermal unfolding of the polypeptide chains.

Notes: The primary receptor (PotF) of the putrescine transport system in E. coli has been crystallized by the hanging-drop vapor-diffusion technique. The crystals belong to the space group P21212 with unit-cell dimensions a = 269.4, b = 82.33 and c = 93.74 Å. The crystals diffract beyond 2.2 Å with a rotating-anode X-ray source. A complete data set from the native crystals has been collected and processed at 2.3 Å resolution. Two heavy-atom derivatives have been prepared from the same Pt compound at 293 and 277 K. The difference Patterson maps revealed completely different major heavy-atom sites between these two derivatives.

Notes: The histidine-containing phosphotransfer (HPt) domain at the C-terminus of the anaerobic sensor kinase ArcB has been cocrystallized with the chemotaxis response regulator CheY by a hanging-drop vapor-diffusion method. Crystals belong to space group P212121 with unit-cell dimensions a = 55.32, b = 76.29 and c = 83.89 Å, with one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.7 Å resolution. This is the first crystallization of a protein–protein complex formed by a transmitter domain of sensor kinase and a receiver domain of response regulator in the two-component signal-transduction system.

Notes: Crystals of the monomeric and dimeric forms of human neutrophil gelatinase associated lipocalin have been grown in hanging-drop vapor-diffusion trials using PEG as a precipitating agent with recombinant protein expressed in a baculovirus-based system. Crystals of monomeric NGAL belong to the cubic space group P432 with lattice constants a = b = c = 126.6 Å; crystals of dimeric NGAL belong to the tetragonal space group P41212 (or its enantiomorph P43212) with lattice constants a = b = 54.14 and c = 121.56 Å. Isomorphous crystals of the NGAL dimer can be grown in the presence of ligand: the tripeptide N-formyl-Met-Leu-Phe.

Notes: Recent work has shown that Scilla campanulata agglutinin from bluebell bulbs has a strong affinity for α(1,3)- and α(1,6)-linked mannosyl residues and possesses moderate antiretroviral activity. This lectin has been crystallized by the hanging-drop method of vapour diffusion complexed with the disaccharide mannose-α1,6-D-mannose. The crystals are in the space group P21212 with unit-cell dimensions a = 70.63, b = 92.79 and c = 47.25 Å, and with a dimer in the asymmetric unit. The crystals diffract X-rays to beyond 1.5 Å resolution at 277 K and are stable in an X-ray beam. Data to 1.6 Å resolution have been collected using a MAR image-plate system at a synchrotron source and the structure of the complex has been solved by the molecular replacement method.

Notes: Profilin is an actin-monomer binding protein that regulates the distribution and dynamics of the actin cytoskeleton. Profilin binds poly-L-proline and proline-rich peptides in vitro and co-localizes with proline-rich proteins in focal adhesions and at the site of actin tail assembly on the surface of intracellular parasites such as Listeria monocytogenes. The crystallization of the complex between human platelet profilin (HPP) and an L-proline decamer [(Pro)10] is reported here. Diffraction from these crystals is consistent with the space group P21212 with unit-cell constants a = 68.25, b = 97.64, c = 39.10 Å. The crystals contain two HPP molecules per asymmetric unit and diffract to 2.2 Å.

Notes: Functional monomeric 83 kDa sialidase L, a NeuAcα2→3Gal-specific sialidase from Macrobdella leech, was expressed in Escherichia coli and readily crystallized by a macroseeding technique. The crystal belongs to space group P1 with unit-cell parameters a = 46.4, b = 69.3, c = 72.5 Å, α = 113.5, β = 95.4 and γ = 107.3°. There is one molecule per unit cell, giving a Vm = 2.4 Å3 Da−1 and a solvent content of 40%. Native and mercury-derivative data sets were collected to 2.0 Å resolution. Threading and molecular-replacement calculations confirmed the existence of a bacterial sialidase-like domain.

Notes: The catalytic domain of the CeIF processive endocellulase, a family 48 glycosyl hydrolase from Clostridium cellulolyticum has been crystallized in the presence of a newly synthesized inhibitor (methyl 4-S-β-cellobiosyl-4-thio-β-cellobioside), by vapour diffusion, using PEG as a precipitant. The protein crystallizes in the orthorhombic P212121 space group and diffracts to a resolution of 2.0 Å. The unit-cell parameters are a = 61.4, b = 84.5, c = 121.9 Å.

Notes: Crystals of the human MHC-encoded molecule MICA, a homologue of MHC class I proteins, have been grown in hanging-drop vapor-diffusion trials using ammonium sulfate as a precipitating agent with recombinant protein expressed in a baculovirus-based system. Cryo-preserved crystals of MICA belong to the cubic space group F4132 with lattice constants a = b = c = 260.7 Å and diffract to a resolution limit of 3.0 Å when cryo-preserved. These crystals do not diffract when handled conventionally.

Notes: The thermostable catechol 2,3-dioxygenase of Bacillus stearothermophilus has been crystallized. The crystal is probably in the space group I222 with unit-cell dimensions of a = 70.87, b = 74.60 and c = 133.69 Å. A native data set has been collected with a completeness of 96% at 2.22 Å resolution and an Rmerge value of 0.091.

Notes: Thiaminase I (Mr = 42 100) from B. thiaminolyticus, expressed in E. coli, has been crystallized by the vapor-diffusion method. Three crystal forms, two of which grew from 0.1 M sodium acetate (pH = 4.6), 0.2 M ammonium sulfate and 30%(w/v) PEG 2000, have been examined by X-ray analysis. One crystal form diffracted to 2.5 Å at room temperature, was orthorhombic, and had unit-cell edges of a = 87.7, b = 120.5 and c = 76.7 Å with space group P212121. A self-Patterson map showed a strong peak indicating noncrystallographic translational pseudosymmetry with (u, v, w) = (0.03, 0.0, 0.5). When these crystals were frozen at liquid-nitrogen temperatures, a second crystal form was observed which had unit-cell dimensions a = 85.5, b = 117.5 and c = 36.6 Å with space group P21212. A third crystal form grew from 0.1 M Tris (pH = 8.5), 0.2 M sodium acetate trihydrate and 28%(w/v) PEG 6000 to produce orthorhombic crystals of space group P212121 with cell edges of a = 114.4, b = 123.1 and c = 92.5 Å.

Notes: A new crystal form of the vanadium-dependent bromoperoxidase from Corallina officinalis has been obtained. The crystals exhibit a `teardrop' morphology and are grown from 2 M ammonium dihydrogen phosphate pH 5 and diffract to beyond 1.7 Å resolution. They are in tetragonal space group P4222 with unit-cell dimensions of a = b = 201.9, c = 178.19 Å, α = β = γ = 90°. A 2.3 Å resolution native data set has been collected at the Hamburg Synchrotron. A mercury derivative data set has also been collected, and the heavy-atom positions have been determined. The self-rotation function and the positions of the heavy atoms are consistent with the molecule being a dodecamer with local 23 symmetry.

Notes: Human glutathione-S-transferase M2–2 (hGSTM2–2) was expressed in Escherichia coli and purified by GSH-affinity chromatography. The recombinant enzyme and the protein isolated from human tissue were indistinguishable based on physicochemical, enzymatic and immunological criteria. The catalytically active dimeric hGSTM2–2 was crystallized without GSH or other active-site ligands in two crystal forms. Diffraction from form A crystals extends to 2.5 Å and is consistent with the space group P21 (a = 53.9, b = 81.5, c = 55.6 Å, β = 109.26 Å) with two monomers in the asymmetric unit. Diffraction from form B crystals extends to 3 Å and is consistent with a space group P212121 (a = 57.2, b = 80.7, c = 225.9 Å) with two dimers in the asymmetric unit. This is the first report of ligand-free mu-class GST crystals, and a comparison with liganded complexes will provide insight into the structural consequences of substrate binding which are thought to be important for catalysis.

Notes: A procedure for the crystallization of recombinant cytochrome P450cam has been developed which avoids the difficulties inherent in the glass-capillary free-interface diffusion method reported previously. The surface mutation, Cys334→Ala (C334A), originally designed to prevent dimer formation and thus improve routine handling of the enzyme, facilitates crystallization by the hanging-drop vapour-diffusion technique. Crystals of (C334A)P450cam grow within 48 h and diffract to beyond 1.2 Å at 100 K in-house on a Siemens multiwire area detector. Data have been collected from the camphor-bound form to 1.35 Å.

Notes: Extended delineation of water molecules, monitored using Rfree values, afforded considerable improvement in quality of electron-density maps for structure determination of mammalian class I and E. coli class II aldolases. Augmented solvent definition results in an additional decrease in Rfree values of 3–4% and is reflected in significantly enhanced electron-density envelopes enabling tracing of amino-acid sequences through regions of otherwise discontinuous or weak electron density.

Notes: The NADP-dependent β-keto acyl carrier protein reductase (BKR) from E. coli has been crystallized by the hanging-drop method of vapour diffusion using poly(ethylene glycol) of average molecular weight 1450. The crystals belong to the hexagonal space group P6122 or P6522 with unit-cell dimensions a = b = 67.8, c = 355.8 Å. Calculated values for Vm and consideration of the packing suggest that the asymmetric unit contains a dimer. BKR catalyses the first reductive step in the elongation cycle of fatty-acid biosynthesis. It shares extensive sequence homology with the enzyme which catalyzes the second reductive step in the cycle, enoyl acyl carrier protein reductase (ENR), and thus provides an opportunity to study the evolution of enzyme function in a metabolic pathway. The structure determination will permit the analysis of the molecular basis of its catalytic mechanism and substrate specificity.

Notes: Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells. These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell. The extracellular region of a p58 KIR specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified. The recombinant KIR2 has been crystallized in 9–10% poly(ethylene glycol) methyl ether (average Mr = 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-β-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method. Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6122 or P6522) with lattice constants a = b = 95.3, c = 130.8 Å. A native data set (3 Å resolution) has been collected at the Photon Factory (λ = 1.0 Å).

Notes: HORF6 is a member of the novel antioxidant enzyme family found in humans. A recombinant form of hORF6 expressed and purified from E. coli has been crystallized by the hanging-drop method using various PEG's as precipitating agents. HORF6 crystallizes in two different monoclinic space groups, P21 and C2. The P21 crystals have unit-cell dimensions of a = 47.85, b = 75.17, c = 63.30 Å and β = 110.21° and contain two monomers per asymmetric unit, while the C2 crystals have unit-cell dimensions of a = 165.27, b = 95.44, c = 166.44 Å and β = 128.97° and contain more than six monomers per asymmetric unit. The P21 crystals with the smaller unit cell diffract X-rays better and behave well for the X-ray analysis. A native data set from a single crystal of the P21 space group gas been collected to 2.0 Å resolution.

Notes: Porphobilinogen synthase (PBGS) catalyzes the condensation of two identical substrate molecules, 5-aminolevulinic acid (ALA), in an asymmetric manner to form porphobilinogen. E. coli PBGS is an homooctameric enzyme. The number of active sites is not clear, but each subunit binds one ZnII ion and one MgII ion. Diffraction-quality crystals of native E. coli PBGS have been obtained, and unit-cell dimensions (a = 130.8, c = 144.0 Å) are reported. These crystals diffract to about 3.0 Å resolution.

Notes: Bowman–Birk trypsin inhibitor from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is tetragonal, belonging to the space group P41212 (or P43212), with unit cell parameters of a = b = 62.48 and c = 94.63 Å. The asymmetric unit contains one molecule of Bowman–Birk trypsin inhibitor with corresponding crystal volume per protein mass (Vm) of 2.89 Å3 Da−1 and the solvent content of 57% by volume. The crystals diffract to at least 1.9 Å Bragg spacing upon exposure to synchrotron X-rays. X-ray data to 1.9 Å have been collected from a native crystal.

Notes: Crystals of the α-hemolysin heptamer, a transmembrane pore-forming toxin from Staphylococcus aureus, have been grown using the nonionic detergents n-decyldimethyl- and n-decyldiethylphosphine oxide, phosphorus homologs of the ionic amine oxide detergents. Five crystal forms were obtained, one of which diffracted X-rays to 3 Å resolution. This crystal form displayed elements of pseudo mm symmetry in screened precession photographs yet it was triclinic with unit-cell dimensions a = 173, b = 173, c = 102 Å, α = 92.6, β = 94.8, γ = 90.2°.

Notes: The NAD+-dependent phenylalanine dehydrogenase from Nocardia sp239 has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. Two crystal forms were obtained in the presence and absence of the enzyme substrates phenylpyruvic acid or phenylalanine and its coenzyme NADH. Crystals of the native protein belong to the hexagonal system, with the space group being one of the enantiomorphic pair P6122 or P6522. The cell dimensions are a = b = 111.0, c = 174.5 Å, α = β = 90 and γ = 120°. Crystals grown from the protein co-crystallized with its substrates all belong to the trigonal system, space group P3121 or P3221, with unit-cell dimensions of a = b = 88.1 , c = 112.6 Å, α = β = 90 and γ = 120°. Preliminary protein-sequencing experiments have established that this enzyme is related to the octameric PheDH's which are members of the wider superfamily of amino-acid dehydrogenases. However, gel-filtration studies suggest that this enzyme is active as a monomer. The full determination of the three-dimensional structure of this phenylalanine dehydrogenase will add to the understanding of the molecular basis of the differential substrate specificity within this enzyme superfamily. In turn this will contribute to the rational design of an amino-acid dehydrogenase which could be used for the diagnosis of phenylketonuria and for the chiral synthesis of high-value pharmaceuticals.

Notes: Allophycocyanin from red alga Porphyra yezoensis has been crystallized in three crystal forms. Form 1 crystals with space group P4132 or P4332 and cell parameters a = 286 Å, α = 90° were obtained by using isopropanol as precipitant. These crystals did not diffract beyond 5.8 Å and could not be used in structure analysis. Form 2 crystals were obtained when crystallization conditions were slightly changed by adding 0.2 M magnesium chloride. The space group of form 2 was not determined because it was difficult to get large single crystals. Form 3 crystals were obtained by using ammonium sulfate as precipitant. The space group of form 3 is R32 with cell dimensions a = b = 105.3, c = 189.4 Å and one \alpha\beta unit in the asymmetric unit. These crystals diffract up to 2.06 Å resolution and are suitable for structure determination by molecular-replacement methods.

Notes: Pentaerythritol tetranitrate (PETN) reductase of Enterobacter cloacae PB2, a flavoprotein involved in the biodegradation of the explosive PETN, ethylene glycol dinitrate (EGDN) and glycerol trinitrate (GTN), was purified from an overexpressing strain of E. coli and crystallized at 293 K using the sitting-drop vapour-diffusion method. Diffraction data can be seen at 1.8 Å. The primitive orthorhombic cell has a monomer in the asymmetric unit. Preliminary molecular-replacement calculations have been performed using a search model based on Old Yellow enzyme.

Notes: Recombinant Sulfolobus solfataricus glyceraldehyde-3-phosphate dehydrogenase has been purified and found to be a tetramer of 148 kDa. The enzyme shows dual cofactor specificity and uses NADP+ in preference to NAD+. The sequence has been compared with other GAPDH proteins including those from other archaeal sources. The purified protein has been crystallized from ammonium sulfate to produce crystals that diffract to 2.4 Å with a space group of P43212 or P41212. A native data set has been collected to 2.4 Å using synchrotron radiation and cryocooling.

Notes: The crystal structure of the zinc-containing exopeptidase bovine carboxypeptidase A (CPA) has been refined to high resolution, based on a data set collected from a single crystal, incorporating new sequence information based on cloning of the bovine gene. In addition, new refined structures are available for the zinc-removed form of the enzyme, apo-CPA, as well as the mercury-replaced form, Hg-CPA. The native structure reveals that the zinc-bound water molecule does not appear to be more loosely bound than the rest of the zinc ligands, at least when B factor values are considered. Nor is there any evidence for a secondary location of this water molecule. The apo-enzyme structure does not show any density in the place of the removed zinc ion. The only significant change appears to be a χ2 rotation of one zinc histidine ligand to form an ion-pair interaction with a glutamic acid side chain. The structure of Hg-CPA reveals a solvent tris molecule bound to the mercury cation, as well as an unidentified cation bound to Glu270. The location of this cation agrees with previous proposals for the binding site of inhibitory zinc. These observations may explain some of the differences in kinetics observed in metal-replaced CPA.

Notes: Crystals of chloroplast NADP-dependent malate dehydrogenase have been grown both with and without the cofactor NADP present. The enzyme has a molecular weight of 43 kDa per subunit and exists as a dimer in solution. The crystals diffract to 2.8 Å and belong to the space group P3221 with cell dimensions a = 148.1, c = 65.5 Å.

Notes: Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme for photosynthesis. The wild-type and mutant (amino-acid substitutions in the catalytically important loop 6 region) enzymes from Chlamydomonas reinhardtii, a unicellular green alga, were crystallized. Wild-type, single-mutant (V331A) and two double-mutant (V331A/T342I and V331A/G344S) proteins were activated with cofactors CO2 and Mg2+, complexed with the substrate analog 2′-carboxyarabinitol-1,5-bisphosphate, and crystallized in apparently isomorphous forms. Unit-cell determinations have been completed for three of the enzymes. They display orthorhombic symmetry with similar cell parameters: wild type a = 130.4, b = 203.3, c = 208.5 Å; single mutant (V331A) a = 128.0, b = 203.0, c = 207.0Å; and double mutant (V331A/T342I) a = 130.0, b = 202.1, c = 209.7 Å. Crystals of the wild-type and single-mutant (V331A) enzymes diffracted to \sim2.8 Å. A small crystal of the double-mutant (V331A/T342I) enzyme diffracted to \sim6 Å. A partial data set (68% complete) of the wild-type protein has been collected at room temperature to about 3.5 Å.

Notes: The water-soluble portion of rat heme oxygenase-1 which lacks 22 hydrophobic amino-acid residues at the C-terminus was expressed in E. coli and crystallized in the form of a complex with heme by the vapor-diffusion method using polyethylene glycol 4000 as the precipitant. The crystals belong to the tetragonal space group P41212 or P43212, with unit-cell dimensions of a = b = 56.7, c = 186.7 Å. The crystal contains one heme–heme oxygenase-1 complex in an asymmetric unit and diffracts X-rays beyond 3.0 Å resolution with a conventional X-ray source.

Notes: Erabutoxin a has been crystallized in its monomeric and dimeric forms. The structures were refined at 1.50 and 1.49 Å resolution, respectively, using synchrotron radiation data. The crystals belong to space group P212121, with cell dimensions a = 49.84, b = 46.62, c = 21.22 Å for the monomer and a = 55.32, b = 53.54, c = 40.76 Å for the dimer. Using starting models from earlier structure determinations, the monomeric structure refined to an R value of 16.7% (8004 unique reflections, 17.0–1.50 Å resolution range), while the dimeric structure has been solved by the molecular-replacement method with a final R value of 16.9% (19444 unique reflections, 17.4–1.49 Å resolution range). The high-resolution electron-density maps clearly revealed significant discrete disorder in the proteins and allowed an accurate determination of the solvent structure. For the monomer, the side chains of six residues were modelled with alternate conformers and 106 sites for water molecules and one site for a sulfate ion were included in the final model, whereas for the dimer, 206 sites for water molecules were included and both C-terminal residues together with the side chains of 11 residues adopted alternative conformations. A comparison was made with earlier structure determinations. The features of the solvent structure of the erabutoxin molecules are discussed in detail.

Notes: Glutamate decarboxylase (GAD) is a vitamin B6 enzyme which catalyzes the α-decarboxylation of L-glutamate to γ-aminobutyric acid (GABA). Escherichia coli cells coexpress two highly homologous enzyme isoforms, GADα and GADβ. Well diffracting crystals of GADβ were obtained by taking advantage of the possibility of expressing each isoform separately. They belong to space group P31 or P32 with the unit-cell dimensions a = b = 115.6 and c = 206.6 Å and contain one GAD hexamer in the asymmetric unit. High-resolution synchrotron data were collected at 100 K for the native protein and a potential heavy-atom derivative.

Notes: Crystals of alcohol dehydrogenase from Sulfolobus solfataricus were grown in the Advanced Protein Crystallization Facility during the Life and Microgravity Sciences Spacelab mission on the US Space Shuttle. Large diffracting crystals were obtained by dialysis, whereas only poor-quality crystals were obtained by vapour diffusion. The quality of both the microgravity and ground-based crystals was analysed by X-ray diffraction. There was some improvement in terms of size and diffraction resolution limit for the microgravity crystals. However, the twinning observed in the Earth-grown crystals was also present for those grown in microgravity.

Notes: A method for locating heavy atoms in the unit cell of macromolecular crystals by a full-symmetry translation function is described. The approach has been implemented in the program TRAHALO and tested on experimental isomorphous and anomalous data.

Notes: Recombinant aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8, has been crystallized in two different crystal forms. The crystals of both forms are orthorhombic and belong to space group P212121 with cell dimensions a = 124.3, b = 113.6 and c = 61.6 Å for form I and a = 197.3, b = 109.7 and c = 80.3 Å for form II. The crystals of form I and II diffract to 2.1 and 2.5 Å resolution, respectively, on a conventional laboratory rotating-anode source. Two heavy-atom derivatives have been identified for form I.

Notes: Creatine kinase (E.C. 2.3.7.2) is an important enzyme in energy metabolism which catalyzes the reversible transfer of a phosphoryl group between phosphocreatine and ADP to give ATP. Large quantities of a brain-type creatine kinase have been isolated from bovine photoreceptor cells and crystals suitable for X-ray diffraction analysis have been obtained by hanging-drop vapor diffusion. Crystals grow as tetragonal bipyramids in space group P43212 with cell dimensions a = b = 96.49, c = 108.42 Å and diffract to at least 2.7 Å resolution.

Notes: The plant aspartic proteinase cardosin A was crystallized using vapour diffusion. Crystals belong to the monoclinic space group C2, cell dimensions a = 116.9 (2), b = 87.2 (8), c = 81.3 (1) Å, β = 104.4 (4)°, and contain two molecules in the asymmetric unit related by a non-crystallographic twofold axis. Diffraction data were collected at room temperature with radiation from a synchrotron source up to 2.85 Å resolution. When the crystals were flash cooled to 110 K in a nitrogen stream the same resolution limit could also be obtained on a rotating-anode source. Recently, synchrotron radiation together with flash cooling led to an improvement of the diffraction data to 1.72 Å resolution.

Notes: The trimeric signal-transduction protein GlnK, from Escherichia coli, has been over-expressed, purified to homogeneity and crystallized. The crystals belong to space group P213 with a = 85.53 Å and have two subunits in the asymmetric unit. The complex of GlnK with ATP crystallized in space group P63 with a = 57.45 Å and c = 54.79 Å. These crystals have a single subunit in the asymmetric unit. High-quality diffraction data from crystals of GlnK and the GlnK complex have been collected to 2.0 Å.

Notes: The hyperthermostable DNA polymerase from a marine Thermococcus archaeon has been crystallized in space group P212121, with unit-cell dimensions a = 94.8, b = 98.2, c = 112.2 Å with one molecule per asymmetric unit. Conditions for data collection at 98 K have been identified, and a complete data set was collected to 2.2 Å resolution. Strategies employed here may facilitate crystallization of other hyperthermostable proteins. The structure of this enzyme will provide the first structural data on the archaeal and hyperthermostable classes of DNA polymerases. Sequence homology to human polymerase \alpha (polymerase B family) may make it a model for studying eukaryotic and viral polymerases and for the development of anti-cancer and anti-viral therapeutics.

Notes: Crystallization of the hydrophilic ectodomain of the epidermal growth factor (EGF) receptor has been accomplished in the presence of the ligand EGF. Two different crystal forms have been obtained, one of which was suitable for X-ray analysis. The space group of this form has been assigned to P21212 with unit-cell dimensions of a = 207.4, b = 113.3 and c = 120.4 Å. A native data set has been recorded and a heavy-atom search is currently under way. Diffraction from these crystals, however, is limited to low resolution and extensive trials to improve crystal quality further have all failed. To analyse the molecular shape and aggregation of the receptor protein in solution, small-angle X-ray diffraction and dynamic light-scattering techniques have been applied. Synchrotron radiation in combination with cryo-techniques is essential for data collection because of the high solvent content and radiation sensitivity.

Notes: The thermophilic and thermostable family B DNA polymerase from the archaeon Sulfolobus solfataricus (Mr of about 100 kDa) has been crystallized by the hanging-drop vapour-diffusion method at 294 K using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 Å and β = 107.8° and diffract up to 2.7 Å resolution on a rotating-anode X-ray source. Native data have been collected at 100 K. A heavy-atom derivative search is in progress.

Notes: Meningococcal meningitis is a severe childhood disease which often results in significant disability or death. Two major etiological agents of meningitis are the group B meningococci and capsular type K1 E. coli. The virulence of these organisms is attributable to structural mimicry between their common α(2–8)-polysialic acid capsular polysaccharide and human tissue antigens, which allows the bacteria to evade immune surveillance. There is currently no effective vaccine to protect against this infection. It has been demonstrated that the capsular polysaccharide of the bacteria can adopt a unique `antigenic conformation'. This antigenic conformation has formed the basis for the development of an N-propionylated polysialic acid vaccine. Immunization trials in mice with this vaccine show the production of two groups of antibodies, of which only N-propionylated polysialic acid-specific were protective. Knowledge of the structure of the antigen-binding site which recognizes the protective epitope is essential to determining the antigenic conformation of the polysaccharides, and is a critical aspect in understanding and improving the action of potential vaccines. The antigen-binding fragments (Fab) of one protective (13D9) and one non-protective (6B9) monoclonal antibody specific for the capsular polysaccharides of group B meningococci have been crystallized and have undergone preliminary X-ray diffraction analysis. Both crystals are observed to scatter X-rays to approximately 1.7 Å resolution at the A1 station at the Cornell High-Energy Synchrotron Source. 13D9 has an orthorhombic unit cell with a = 41.8, b = 102.3, c = 134.7 Å, with space group P212121. Fab 6B9 has an orthorhombic unit cell with a = 89.6, b = 132.0 and c = 36.9 Å, with space group P21212.

Notes: Portal proteins are cyclical oligomers which play essential roles in bacteriophage pro-capsid formation, DNA packaging, and in connector formation. Bacteriophage SPP1 portal protein (gp6) is a turbine-like molecule with 13-fold symmetry [Dube et al. (1993) EMBO J. 12, 1303–1309]. The purified protein was crystallized with polyethylene glycol 400 as the precipitating agent using the vapor-diffusion method. Salt conditions were selected based on the properties of gp6 in different ionic environments. X-ray diffraction data up to a resolution of 7.85 Å were measured from frozen crystals with orthorhombic space group C2221 and cell dimensions a = 180.5 (5), b = 223.5 (5), c = 417 (1) Å. The asymmetric unit contains one tridecameric portal protein with 57.3 kDa subunits. The self-rotation searches confirm the 13-fold symmetry of the crystallized protein.

Notes: Pap1 is a fission yeast transcription factor that activates genes related with resistance against staurosporine, a potent inhibitor of protein kinase C, and has been shown to be involved in cell growth, cell cycle, carcinogenesis and differentiation. Pap1 has the bZIP DNA-binding domain but binds to non-consensus DNA sequences for the bZIP motif. Highly ordered crystals of the DNA-binding domain complexed with a DNA fragment that has an ATF/CREB-like non-consensus sequence have been obtained. The crystals grew by the vapor-diffusion technique with polyethylene glycol 6000 and belong to space group R3 with a = b = 240.78, c = 43.85 Å. A 2.0 Å resolution data set was collected with a cryo-crystallographic technique.

Notes: Recombinant citrate synthase from a psychrotolerant bacterium, DS2–3R, recently isolated in Antarctica, has been crystallized. The crystals belong to space group P6122 or P6522, with cell dimensions a = b = 70.8, c = 307.8 Å. Diffraction data collected on a synchrotron from a cryoprotected crystal extend to at least 2.0 Å. Knowledge of the structure of this enzyme will add to the understanding of cold activity and thermolability, and will be of biotechnological interest. Previously, the structure of citrate synthase from Archaea inhabiting environments at 328 and 373 K, has been reported. This present study will extend our understanding of the structural integrity and activity of proteins at the temperature extremes of life.

Notes: The α-toxin of Clostridium perfringens is the major virulence determinant for gas gangrene in man. The gene encoding the α-toxin has been cloned into E. coli from two strains of the bacterium (NCTC8237 and CER89L43) and subsequently purified to homogeneity. The two strains of α-toxin differ by five amino acids, resulting in the toxin from NCTC8237 being sensitive to chymotrypsin digestion while that from CER89L43 is resistant. The α-toxin from each of these strains has been crystallized in two different forms by the hanging-drop vapour-diffusion method at 293 K. CER89L43 form I crystals belong to space group R32 and have two molecules in the crystallographic asymmetric unit and a unit cell with a = b = 151.4, c = 195.5 Å, α = β = 90, γ = 120°. The crystals diffracted to dmin = 1.90 Å. The characteristics of the NCTC8237 form I crystals have already been reported. The form II crystals from both strains belong to space group C2221 with one molecule in the crystallographic asymmetric unit and, for strain CER89L43, have cell dimensions a = 61.05, b = 177.50, c = 79.05 Å, α = β = γ = 90°, while for strain NCTC8237 the cell dimensions are a = 60.50, b = 175.70, c = 80.20 Å, α = β = γ = 90°. The crystals diffracted to maximum resolutions of 1.85 and 2.1 Å for the CER89L43 and the NCTC8237 strains, respectively.

Notes: Chitinase is necessary for fungal growth and cell division and, therefore, is an ideal target for the design of inhibitors which may act as antifungal agents. A chitinase from the fungal pathogen Coccidioides immitis has been expressed as a fusion protein with gluathione-S-transferase (GST), which aids in purification. After cleavage from GST, chitinase was crystallized from 30% PEG 4000 in 0.1 M sodium acetate pH 4.6. The crystals have a tetragonal crystal lattice and belong to space group P41212 or P43212 and diffract to 2.2 Å resolution. The unit-cell parameters are a = b = 91.2, c = 95.4 Å; there is only one chitinase molecule in the asymmetric unit.

Notes: Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic crystals belong to space group P21 with cell dimensions a = 79.1, b = 89.2, c = 145.4 Å and β = 92.8°, and diffract to at least 2.6 Å. The orthorhombic crystals belong to space group P21212 with a = 89.4, b = 155.9, c = 79.5 Å, and diffract to 2.9 Å. Diffraction from the hexagonal form showed extensive disorder. The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centring. The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form. Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to the understanding of protein thermostability.

Notes: Polyamine oxidase catalyses the oxidation of the secondary amino group of spermine, spermidine and their acetyl derivatives. The enzyme plays an important role in the regulation of polyamine intracellular concentration and is a member of the family of flavin-containing amine oxidases. Crystals of maize polyamine oxidase have been grown by the hanging-drop vapour-diffusion technique. The crystals are in hexagonal space group P6122 (or P6522) with cell dimensions a = b = 184.6, c = 280.9 Å. A native data set has been collected to 2.7 Å resolution at a synchrotron radiation source.

Notes: Two forms of the archaeal intron-encoded site-specific endonuclease I-DmoI, namely I-DmoIc and I-DmoIl, have been purified and crystallized. Crystals of I-DmoIc are rod-shaped and diffract to 3.0 Å resolution, but further analysis was hampered by twinning. Crystals of I-DmoIl, which is a six-amino-acid C-terminal truncation of I-DmoIc, are plate shaped and belong to space group C2 with cell parameters a = 93.72, b = 37.03, c = 55.56 Å, β = 113.4°, with one molecule per asymmetric unit (Vm = 2.01 Å3 Da−1). The crystals diffract to at least 2.3 Å resolution. A complete native data set has been measured and structure determination is on-going.

Notes: The phospholipases A2 (PLA2, E.C. 3.1.1.4, phosphatide sn2 acylhydrolases) are the major components of the venom of several snakes. They are responsible for several important pharmacological effects observed in ophidian incidents. PLA2 piratoxin II from Bothrops pirajai has been crystallized by the vapour-diffusion technique. X-ray diffraction data have been collected to 2.04 Å resolution (90.2% complete, Rmerge = 0.070). The space group is P21 and the cell parameters are a = 46.19, b = 60.36, c = 58.74 Å and β = 96.05°. The structure has been solved by molecular replacement using the crystallographic structure of PLA2 from Bothrops asper (PDB code 1CLP) as a search model.

Notes: The seed lectin DBL and the related stem and leaves lectin DB58 of the tropical legume Dolichos biflorus were crystallized, as well as complexes of DBL with adenine and with GalNAc(α1-3)[Fuc(α1-2)]Gal. The different crystal forms of DBL diffract to about 2.8 Å, while DB58 crystals diffract to 3.3 Å.

Notes: GerE is the latest-acting of a series of factors which regulate gene expression in the mother cell during sporulation in Bacillus. The gene encoding GerE has been cloned from B. subtilis and overexpressed in Escherichia coli. Purified GerE has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. The small plate-like crystals belong to the monoclinic space group C2 and diffract beyond 2.2 Å resolution with a synchrotron radiation X-ray source.

Notes: PhoB is the response regulator of the E. coli two-component signal transduction system for phosphate regulation. It is a transcription factor that activates more than 30 genes of the pho regulon. Crystals of the receiver domain of PhoB were obtained by applying the hanging-drop vapour-diffusion method. X-ray diffraction data have been collected using synchrotron radiation to 1.88 Å resolution. The crystals belong to the orthorhombic space group P212121 with unit-cell constants a = 34.11, b = 60.42, c = 119.97 Å. The Matthews parameter suggests that PhoB crystallizes with two molecules per asymmetric unit, suggesting that activating dimerization occurs in the crystal.

Notes: The structure of Nudaurelia capensis ω virus (NωV), a single-stranded RNA virus, was determined to 2.8 Å resolution. Triclinic crystals (a = 413.6, b = 410.2, c = 419.7 Å, α = 59.13, β = 58.9, γ = 64.0°) diffracted X-rays beyond 2.7 Å resolution. The unit cell contained one icosahedral virus particle, providing 60-fold non-crystallographic symmetry (n.c.s.) and structural redundancy. The particle orientation in the unit cell was determined by self-rotation function analyses. Initial phases to 18 Å resolution were derived from a hollow spherical model of 192 Å outer radius and 139 Å inner radius, filled with uniform electron density. Radii of the model were determined by maximizing the correlation of the model-based calculated data with the low-resolution X-ray diffraction and solution-scattering data. Phases were refined by 60-fold non-crystallographic electron-density averaging and extended in small steps to a resolution of 5 Å. The phases obtained represented a mixture of four different phase sets, each consistent with the icosahedral symmetry constraints. The resulting electron density was not interpretable. A difference Fourier map computed with the native and an isomorphous heavy-atom derivative data sets and phases refined by real-space averaging was interpretable only if data within the 10 Å resolution shell were used. Maps calculated with data significantly higher than 10 Å resolution failed to display a constellation of heavy-atom sites consistent with the T = 4 icosahedral symmetry. Attempts to extend the phases beyond 10 Å resolution, starting with either phases based on a model or single isomorphous replacement, were unsuccessful. Successful phase extension was achieved by computing the phases for the higher resolution reflections from a partial atomic model (poly gly) built into the averaged 10 Å electron-density map. Phases from this model served as the starting point for n.c.s. phase refinement and extension to slightly higher resolution. The atomic model was improved at each extension interval and these phases were used for the subsequent phase calculation and extension. The entire polypeptide backbone corresponding to the NωV structure was built into the map at 4 Å. The same procedure for phase refinement was used to extend the phases to 2.8 Å in small increments of resolution. The overall molecular averaging R factor and correlation coefficient at 2.8 Å resolution were 18.4% and 0.87, respectively.

Notes: Tyrosine aminotransferase from Trypanosoma cruzi has been crystallized from PEG 4000 at pH 6.8. The crystals belong to the monoclinic space group P21 and have lattice constants of a = 59.1, b = 103.0, c = 77.8 Å, β = 113.1° for a data set measured at 138 K. The presence of a non-crystallographic twofold axis together with a Matthews parameter Vm of 2.5 Å3 Da−1 indicates that the asymmetric unit contains one dimeric molecule. The crystals diffract to at least 2.7 Å and are stable in the X-ray beam in a shock-frozen state. Native data sets have been collected at temperatures of 285 and 138 K using a Siemens X1000 detector on a rotating-anode generator.

Notes: Quinolinate phosphoribosyltransferase (QPRT), purified from hog liver, has been crystallized using PEG 8000 as the precipitant. The crystals form long hexagonal rods in the space group P6322 with cell dimensions a = b = 121.7, c = 94.5 Å. Based on the unit-cell dimensions and the calculated molecular mass of 33 500 Da, the Matthews coefficient suggests one molecule per asymmetric unit (Vm = 3.45 Å3 Da−1; 64% solvent). Three native data sets were collected to a resolution of 2.5 Å and merged to provide a set that is 94.7% complete, with an Rsym value of 9.6%.

Notes: The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 Å resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 Å and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.

Notes: The NS3 protein of hepatitis C virus (HCV) is thought to be essential for viral replication. The N-terminal domain of the protein contains protease activity and the C-terminal domain contains nucleotide triphosphatase and RNA helicase activity. The RNA helicase domain of HCV NS3 protein was purified by using affinity-column chromatographic methods, and crystallized by using the microbatch crystallization method under oil at 277 K. The crystals belong to primitive trigonal space group P3121 or P3221 with cell dimensions of a = b = 93.3, c = 104.6 Å. The asymmetric unit contains one molecule of the helicase domain, with the crystal volume per protein mass (Vm) of 2.50 Å3 Da−1 and solvent content of about 50.8% by volume. A native data set to 2.3 Å resolution was obtained from a frozen crystal indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.

Notes: A bleomycin-binding protein (BLMA) produced by bleomycin-producing Streptomyces verticullus was crystallized in a form suitable for X-ray diffraction analysis using the vapor-diffusion method. Crystals were grown at pH 5.7, in 0.2 M NH4 actate and 0.1 M Na acetate, using 30% PEG 4000 as a precipitant. They belong to the orthorhombic system, with space group P21212, cell dimensions a = 54.90, b = 67.94, c = 35.60 Å, and one BLMA molecule in the asymmetric unit. The crystals diffract X-rays well and the diffraction intensity data was collected up to 1.5 Å resolution with a merging R value of 0.054 at beamline 6B of the Photon Factory. The diffraction data set is 94% complete.

Notes: The crystal structure of a pepsin from the gastric mucosa of Atlantic cod has been determined to 2.16 Å resolution. Data were collected on orthorhombic crystals with cell dimensions a = 35.98, b = 75.40 and c = 108.10 Å, on a FAST area-detector system. The phase problem was solved by the molecular-replacement method using porcine pepsin (PDB entry 5PEP) as a search model. The structure has been refined to a crystallographic R factor of 20.8% using all reflections between 8.0 and 2.16 Å, without prior knowedge of the primary sequence. The resulting crystal structure is very similar to the porcine enzyme, consisting of two domains with predominantly β-sheet structure in the same sequential positions as the enzyme from pig. In the course of the model building, 122 residues were substituted and two residues deleted from the starting model to give a polypeptide chain of 324 amino acids and a sequence identity of 57.7% with the pig pepsin. No carbohydrate residues were located. Sequence alignment with available aspartic proteinases, indicates that the fish enzyme seems to be more related to mammalian gastric pepsins than to the mammalian gastricsins and chymosins, lysosomal cathepsin D's and a pepsin from tuna fish. The amino-acid composition of the cod enzyme, however, is more in accordance with the cathepsin D's.

Notes: The X-ray structure of the 36 kDa soluble lytic transglycosylase from Escherichia coli has been determined starting with the multiple isomorphous replacement method with inclusion of anomalous scattering at 2.7 Å resolution. Subsequently, before any model building was carried out, phases were extended to 1.7 Å resolution with the weighted automated refinement procedure wARP, which gave a dramatic improvement in the phases. The electron-density maps from wARP were of outstanding quality for both the main chain and the side chains of the protein, which allowed the time spent on the tracing, interpretation and building of the X-ray structure to be substantially shortened. The structure of the soluble lytic transglycosylase was refined at 1.7 Å resolution with X-PLOR to a final crystallographic R factor of 18.9%. Analysis of the wARP procedure revealed that the use of the maximum-likelihood refinement in wARP gave much better phases than least-squares refinement, provided that the ratio of reflections to protein atom parameters was approximately 1.8 or higher. Furthermore, setting aside 5% of the data for an R_{\rm free} test set had a negative effect on the phase improvement. The mean W_{wARP}, a weight determined at the end of the wARP procedure and based on the variance of structure factors from six individually refined wARP models, proved to be a better indicator than the R_{\rm free} factor to judge different phase improvement protocols. The elongated Slt35 structure has three domains named the alpha, beta and core domains. The alpha domain contains mainly \alpha-helices, while the beta domain consists of a five-stranded antiparallel \beta-sheet flanked by a short \alpha-helix. Sandwiched between the alpha and beta domains is the core domain, which bears some resemblance to the fold of the catalytic domain of the previously elucidated 70 kDa soluble lytic transglycosylase from E. coli. The putative active site is at the bottom of a large deep groove in the core domain.

Notes: The D-enantiomer of a potently sweet protein, monellin, has been crystallized and analyzed by X-ray crystallography at 1.8 Å resolution. Two crystal forms (I and II) appeared under crystallization conditions similar, but not identical, to the crystallization conditions of natural L-monellin. There are four molecules per asymmetric unit in crystal form I and one in crystal form II. Crystal form I is not reproducible and is equivalent to that of monoclinic L-monellin. Intermonomer contacts in crystal form II are very different from those found in natural L-monellin crystals. The backbone trace of D-monellin resembles very closely the mirror image of that of L-monellin, but the N- and C-terminus backbones as well as several side-chain conformations of D-monellin are different from those of natural L-monellin. Most of these apparent differences may be attributable to the crystal packing differences.

Notes: The synchrotron Laue technique has been applied to high-resolution structure refinement of the ribotoxin, restrictocin [Yang & Moffat (1996). Structure, 4, 837–852]. By employing carefully designed data-collection strategies and the data-reduction algorithms incorporated in the software system LaueView [Ren & Moffat (1995a). J. Appl. Cryst. 28, 461–481; Ren & Moffat (1995b). J. Appl. Cryst. 28, 482–493], a set of high-resolution Laue data with a completeness and accuracy comparable to excellent monochromatic data was obtained. Through detailed comparison with the monochromatic data and electron-density maps derived from the Laue data, optimum data-collection and reduction strategies were identified and the application of Laue diffraction techniques to conventional crystallographic refinement was demonstrated.

Notes: The three-dimensional structure and associated solvent of human carboxyhemoglobin at 2.2 Å resolution are compared with other R-state and T-state human hemoglobin structures. The crystal form is isomorphous with that of the 2.7 Å structure of carboxyhemoglobin reported earlier [Baldwin (1980). J. Mol. Biol. 136, 103–128], whose coordinates were used as a starting model, and with the 2.2 Å structure described in an earlier report [Derewenda et al. (1990). J. Mol. Biol. 211, 515–519]. During the course of the refinement, a natural mutation of the α-subunit, A53S, was discovered that forms a new crystal contact through a bridging water molecule. The protein structure shows a significant difference between the α and β heme geometries, with Fe—C—O angles of 125 and 162°, respectively. The carboxyhemoglobin is compared with other fully ligated R-state human hemoglobins [Baldwin (1980). J. Mol. Biol. 136, 103–128; Shaanan (1983). J. Mol. Biol. 195, 419–422] with the R2-state hemoglobin [Silva et al. (1992). J. Biol. Chem. 267, 17248–17256] and with T-state deoxyhemoglobin [Fronticelli et al. (1994). J. Biol. Chem. 269, 23965–23969]. The structure is similar to the earlier reported R-state structures, but there are differences in many side-chain conformations, the associated water structure and the presence and the position of a phosphate ion. The quaternary changes between the R-state carboxyhemoglobin and the R2-state and T-state structures are in general consistent with those reported in the earlier structures. The location of 238 water molecules and a phosphate ion in the carboxyhemoglobin structure allows the first comparison of the solvent structures of the R-state and T-state structures. Distinctive hydration patterns for each of the quaternary structures are observed, but a number of conserved water molecule binding sites are found that are independent of the conformational state of the protein.

Notes: Several real-space indices and temperature factors are compared with respect to their correlation with atomic positional error and their ability to indicate atoms and residues with the worst of subtle errors. The best index, rED, is a correlation coefficient between model and map electron densities, similar to one proposed earlier, but incorporating two improvements. Firstly, resolution is accounted for explicitly by calculating the model electron density by Fourier transformation of resolution-truncated scattering factors. Secondly, the deviation between model and map electron densities is assigned to neighboring atoms according to their contribution to the electron density of each grid point. With maps of various qualities, rED is the single index with best correlation to atomic error with grouped or individual atoms, and it is the most reliable indicator of poor residues. With poorer omit maps, imprecision of individual atoms is best diagnosed by a combination of low rED or high B factor. With the improved methods, 60–70% of the least precise atoms can detected in a fully refined structure. Similarly, 40–80% of the least precise atoms of an unrefined model can be detected by comparison with an isomorphous replacement map. This is useful in assessing and improving the quality of a model, but not sufficient to confidently validate all atoms of a structure at sub-atomic resolution.

Notes: The Fab fragments of two monoclonal antibodies (Fab3A2, Fab6A) raised against epitopes of human chorionic gonadotrophin (hCG) have been crystallized using the vapour-diffusion technique. The Fab3A2 antibody recognises an epitope on the C-terminal peptide of the β-subunit and the Fab6A a conformational epitope of hCG. Both Fab crystals grow as hexagonal rods from ammonium sulfate solutions. The Fab3A2 crystals belong to space group P3121 with a = b = 74.84, c = 198.2 Å and diffract to 1.33 Å at the ESRF. The Fab6A crystals are in the space group P3221 with a = b = 129.53, c = 74.40 Å and diffract to 2.7 Å at the Daresbury SRS. One Fab molecule per asymmetric unit is present in both crystals.

Notes: Amino-acid dehydrogenases catalyse the interconversion of their respective amino acids to the corresponding keto acid, with concomitant reduction of NAD or NADP. The enzymes phenylalanine, glutamate, leucine and valine dehydrogenase all share a similar three-dimensional subunit structure and a high degree of sequence similarity, indicating that they belong to an enzyme superfamily related by divergent evolution. In contrast, alanine dehydrogenase shows no sequence similarity with any of these enzymes despite catalysing a reaction with the same chemistry and thus it is predicted that it possesses a different three-dimensional structure. The alanine dehydrogenase from Phormidium lapideum has been crystallized in space group R32, cell dimensions a = b = 123.1 and c = 184.8 Å, with a monomer in the asymmetric unit. The structure determination of this enzyme will shed light on how nature has evolved two different systems to carry out the same reaction.

Notes: Recombinant homoserine dehydrogenase from Saccharomyces cerevisiae has been crystallized in three different forms. Crystals of the apo-enzyme belong to the tetragonal space group P4 and have unit-cell-dimensions a = b = 130 and c = 240 Å. The resolution limit for these crystals is 3.9 Å. Crystals of homoserine dehydrogenase grown in the presence of the co-factor NAD+ have the tetragonal space group P41212 or its enantiomorph P43212. The unit-cell dimensions for these crystals are a = b = 80.4 and c = 250.2 Å, and the observed resolution limit is 2.2 Å. Protein crystals grown in the presence of the product L-homoserine and the inert NAD+ analogue 3-aminopyridine adenine dinucleotide belong to the monoclinic space group P21 with unit-cell parameters a = 58.8, b = 104.2, c = 120.7 Å, β = 91.9°. This last crystal form has a diffraction limit of 2.7 Å resolution.

Notes: Crystals of an intact GST–estrogen receptor hormone binding domain fusion protein have been grown from solutions of MPD. The crystals grew as clusters of thin plates and needles of maximum dimensions 100 × 20 × 1 µm but were unsuitable for X-ray diffraction analysis. However, examination by electron microscopy shows an ordered lattice in which the protein molecules are clearly visible. Image analysis of electron micrographs of the protein crystals revealed electron stain-excluding density which showed a two-domain trimeric structure in projection, with each molecule of dimensions 12.0 × 5.0 nm diameter. The use of GST-fusion proteins in crystallization are discussed.

Notes: Acetyl xylan esterase is involved in the biodegradation of hemicellulose. It cleaves O-acetyl groups from xylan, which is the most abundant hemicellulose in nature. The catalytic core of acetyl xylan esterase from T. reesei has been crystallized and X-ray diffraction data at 2.3 Å collected. The crystal belongs to the triclinic space group P1 with unit-cell parameters a = 50.3, b = 62.1, c = 40.0 Å, α = 110.1, β = 113.6 and γ = 97.9°. The asymmetric unit contains two molecules.

Notes: Faster workstations with larger memories are making error estimation from full-matrix least-squares refinement a more practicable technique in protein crystallography. Using minimum variance weighting, estimated standard deviations of atomic positions have been calculated for two eye lens proteins from the inverse of a least-squares normal matrix which was full with respect to the coordinate parameters. γB-crystallin, refined at 1.49 Å yielded average errors in atomic positions which ranged from 0.05 Å for main-chain atoms to 0.27 Å for unrestrained water molecules. The second structure used in this work was that of βB2-crystallin refined at 2.1 Å resolution where the corresponding average errors were 0.08 and 0.35 Å, respectively. The relative errors in atomic positions are dependent on the number and kinds of restraints used in the refinements. It is also shown that minimum variance weighting leads to mean-square deviations from target geometry in the refined structures which are smaller than the variances used in the distance weighting.

Notes: In earlier sodium dodecylsulfate–polyacylamide gel electrophoresis (SDS–PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2–0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026–0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS–PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS–PAGE analysis of OHEWL yielded two closely spaced dimer bands with Mr = 28 000 and 27 500. In addition, larger HEWL oligomers with Mr = 1.7 million and 320 000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 µg−1 mg ml−1 OHEWL to 32 mg ml−1 HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03–0.3 M hydrogen peroxide. Repartitioning of the dimer during crystallization at various temperatures between 277 and 293 K was analyzed by SDS–PAGE. The crystals contained ≤ 25%(w/w) of the oligomers in the solution, with no apparent temperature dependence of the repartitioning.

Notes: Azurin from Pseudomonas putida is a blue copper protein which functions as an electron carrier. Two crystal forms of azurin were grown, one in the presence and the other in the absence of zinc acetate; each belongs to space group P21 and contains two molecules per asymmetric unit. The zinc-free crystals have cell dimensions a = 43.25, b = 50.65, c = 54.60 Å, β = 107.79°, while the crystals grown from zinc-containing solution have cell dimensions a = 40.76, b = 51.22, c = 54.96 Å, β = 103.12°. The latter crystals were found to have four zinc ions incorporated into the crystal lattice. Both crystal structures were solved by the molecular-replacement method using the program MERLOT. The search model was the structure of azurin from Alcaligenes denitrificans. The crystallographic R factor for native azurin is 0.169 (Rfree = 0.257) from 8 to 1.92 Å resolution, while that for zinc azurin is 0.181 (Rfree = 0.248) from 10 to 1.6 Å resolution; for each structure the root-mean-square deviation in bond lengths from ideal values is 0.007 Å. In both crystal structures the Cu atom forms three strong bonds in the equatorial plane, two with N^{\delta1} from His46 and His117, and one with the thiolate S atom of Cys112. Two longer axial approaches are made by the S^\gamma from Met121 and the carbonyl O atom from Gly45. This results in a distorted trigonal bipyramidal co-ordination around the Cu atom. It further confirms the presence of a weak fifth bond to the copper in P. putida azurin, as with other azurin structures described at high resolution. The N^{\delta1} atom of His35 is protonated, as it is in the low-pH form of azurin from Pseudomonas aeruginosa but unlike the low-pH form of the azurins from Alcaligenes denitrificans or Alcaligenes xylosoxidans. In each crystal form the two molecules of azurin in the asymmetric unit are related by a local twofold axis and form a dimer stabilized by the interaction of a pair of hydrophobic patches surrounding the partially exposed His117 side chain. In the other known azurin crystal structures, analogous dimer formation is observed, but with different relative orientations of the molecules. The four zinc ions introduced during crystallization of zinc azurin are bound to the protein and participate in five- and sixfold ligand coordination with no affect on the copper binding site. The zinc ligands are N^\delta from His, carboxylate O atoms from Asp and Glu, O^\gamma from Ser and water molecules. One of the zinc ions, located on a non-crystallographic twofold axis, links the dimers of the asymmetric unit into continuous chains parallel to the crystallographic (−101) direction and is primarily responsible for the altered unit-cell parameters. Two of the other zinc ions bind to His83, one in each molecule.

Notes: The basic phospholipase A2 isolated from the venom of Agkistrodon halys Pallas (Agkistrodon blomhoffii Brevicaudus) is a hemolytic toxin and one of the few PLA2's capable of hydrolyzing the phospholipids of E. coli membranes in the presence of a bactericidal/permeability-increasing protein (BPI) of neutrophils. The crystal structure has been determined and refined at 2.13 Å to a R factor of 16.5% (F 〉 3\sigma) with excellent stereochemistry. A superposition of the two molecules in the asymmetric unit gives an r.m.s. deviation of 0.326 Å for all Cα atoms. The refined structure allowed a detailed comparison with other PLA2 species of known structures. The overall architecture is similar to those of other PLA2's with a few significant differences. One of which is in the region connecting the N-terminal helix and the Ca2+-binding loop. Unexpectedly, the conformation of the peptide plane Cys29—Gly30 in the Ca2+-binding loop is very different to that of other PLA2's. The amide NH of Gly30 does not point toward the proposed site for stabilization of the tetrahedral intermediate oxyanion of the substrate analogue. The structure includes four residues which occur less frequently in other PLA2's. His1, Arg6 and Trp70 located at the interfacial recognition site may play an important role in the interaction with aggregated substrates, while Trp77 contributes to the hydrophobic interactions between the β-wing and the main body of the molecule. This structure analysis reveals that two clusters of basic residues are located at or near the interfacial recognition site, forming an asymmetric positively charge distribution. In contrast to the acidic isoform, the present enzyme is a dimer in the crystalline state. The special phospholipid hydrolysis behaviors are discussed in the light of the structure determined.

Notes: The last five years have seen a large increase in the use of cross validation in the refinement of macromolecular structures using X-ray data. In this technique a test set of reflections is set aside from the working set and the progress of the refinement is monitored by the calculation of a free R factor which is based only on the excluded reflections. This paper gives estimates for the ratio of the free R factor to the R factor calculated from the working set for both unrestrained and restrained refinement. It is assumed that both the X-ray and restraint observations have been weighted correctly and that there is no correlation of errors between the test and working sets. It is also shown that the least-squares weights that minimize the variances of the refined parameters, also approximately minimize the free R factor. The estimated free R-factor ratios are compared with those reported for structures in the Protein Data Bank.

Notes: Potential benefits of using short X-ray wavelengths for protein crystal data collection arise from a reduction in absorption errors and a decrease in radiation damage of a sample. On the other hand, at longer wavelengths one may benefit from an increase in scattering efficiency of a crystal and an increase in intensity of an incident beam at a given synchrotron beamline. For small and frozen crystals the negative effects of absorption and radiation damage would be minimized which may shift the balance of interests towards the use of longer wavelengths. Experiments carried out at EMBL beamlines at DESY (Hamburg) show an advantage of using wavelengths longer than 1 Å for data collection from crystals of up to 0.5 mm.

Notes: The structure of rusticyanin, an acid-stable copper protein, has been determined at 2.1 Å resolution by direct methods combined with the single-wavelength anomalous scattering (SAS) of copper (f^{\prime\prime} = 3.9 e−) and then conventionally refined (Rcryst = 18.7%, Rfree = 21.9%). This is the largest unknown protein structure (Mr ∼ 16.8 kDa) to be determined using the SAS and direct-methods approach and demonstrates that by exploiting the anomalous signal at a single wavelength, direct methods can be used to determine phases at typical (∼2 Å) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper (f^{\prime\prime} ∼ 4 e−), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se (f_{\rm max}^{\prime\prime} = 8 e− at the `white line') and 80 kDa for a Pt derivative (f_{\rm max}^{\prime\prime} = 19 e− at the `white line', L3 edge). The method provides a powerful alternative in solving a de novo protein structure without either preparing multiple crystals (i.e. isomorphous heavy-atom derivative plus native crystals) or collecting multi-wavelength anomalous diffraction (MAD) data.

Notes: Human complement component C5 has been crystallized using a low-salt batch technique. The crystals are large hexagonal bi-pyramids often larger than 1.5 mm. Although these crystals were grown in low salt (0.1 M NaCl), they are remarkably stable for at least 2 months at 281 K and they are not dissolved in aqueous buffers containing up to 2 M sodium chloride. The space group is P3121 or P3221, and the cell parameters were determined to be a = 144.9, b = 144.9, c = 243.1 Å; α = 90°, β = 90, γ = 120°. At room temperature and cryo-temperatures the crystals diffract at best to 6 Å using rotating-anode X-ray sources. Using synchrotron radiation with cryoprotection using 40%(v/v) PEG 400 the resolution limit can be extended to 3.3 Å. In both cases the crystals show significant anisotropy, with relatively weaker reflections at higher resolution in the a*b* plane.

Notes: One enzyme which catalyzes the last step of the formation of the hormone retinoic acid from vitamin A (retinol) is retinal dehydrogenase type II (RalDH2). RalDH2, expressed in the Escherichia coli BL21(DE3) strain, was purified and crystallized using ammonium sulfate as a precipitant. These crystals belong to the space group P212121 (a = 108, b = 150, c = 168 Å, α = β = γ = 90°).

Notes: The efficiency of molecular-replacement methods in the structure analysis of B-DNA is markedly increased if a knowledge of the structural properties and helical symmetry of B-DNA is incorporated into molecular-replacement procedures. The separation of the most significant or most robust parameters, such as the location of helices in the unit cell, from the less well defined parameters, such as rotation around the helix axis, further improves the reliability of molecular replacement and avoids frameshift errors in the positioning of the model. This approach has been applied successfully to solve novel structures of four B-DNA decamers in various space groups.

Notes: The 1.89 Å resolution structure of the complex of bovine pancreatic phospholipase A2 (PLA2) with the transition-state analogue L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine (TSA) has been determined. The crystal of the complex is trigonal, space group P3121, a = b = 46.58 and c = 102.91 Å and isomorphous to the native recombinant wild type (WT). The structure was refined to a final crystallographic R value of 18.0% including 957 protein atoms, 88 water molecules, one calcium ion and all 31 non-H atoms of the inhibitor at 1.89 Å resolution. In all, 7 726 reflections [F\gt2\sigma(F)] were used between 8.0 and 1.89 Å resolution. The inhibitor is deeply locked into the active-site cleft and coordinates to the calcium ion by displacing the two water molecules in the calcium pentagonal bipyramid by the anionic O atoms of the phosphate and phosphonate group. The hydroxyl group of Tyr69 hydrogen bonds to the second anionic O atom of the phosphate group while that of the phosphonate group replaces the third water, `catalytic' water, which forms a hydrogen bond to Nδ1 of His48. The fourth water which also shares Nδ1 of His48 is displaced by the steric hinderance of the inhibitor. The fifth conserved structural water is still present in the active site and forms a network of hydrogen bonds with the surrounding residues. The structure is compared with the other known TSA–PLA2 complexes.

Notes: Azurin I from Alcaligenes xylosoxidans NCIMB 11015 (AzN-I) was crystallized by using PEG 4000 as a precipitant. The crystals belong to the monoclinic crystal system and have a space group C2 with the unit-cell parameters of a = 130.67, b = 54.26, c = 74.55 Å, and β = 95.99°. The structure of AzN-I has been solved by the molecular replacement method. Azurin II from the same bacterium (AzN-II) was chosen as the initial structural model. The final crystallographic R value is 17.3% and free R value is 23.6% for 10 958 reflections at a resolution of 2.45 Å. The root-mean-square deviations for main-chain atoms range between 0.19 and 0.26 Å among the four independent molecules in the asymmetric unit. The Cu atom is coordinated to Nδ of His46 and His117 at 2.0 (1) and 1.9 (1) Å, and to Sγ of Cys112 at 2.2 (1) Å, while the carbonyl O atom of Gly45 and Sδ of Met121 coordinate axially to Cu atom at 2.5 (1) and 3.1 (1) Å, respectively. The Cu—N and Cu—S distances of AzN-I are quite similar to those of AzN-II, however, the Cu—O (Gly45) bond length in AzN-I is 0.25 Å shorter than its counterpart in AzN-II. The results have been used to discuss the differences in the spectra of these two proteins.

Notes: The crystal structures of two double mutants (F14N/V21T and F14N/V86T) of the signal transduction protein CheY have been determined to a resolution of 2.4 and 2.2 Å, respectively. The structures were solved by molecular replacement and refined to final R values of 18.4 and 19.2%, respectively. Together with urea-denaturation experiments the structures have been used to analyse the effects of mutations where hydrophobic residues are replaced by residues capable of establishing hydrogen bonds. The large increase in stabilization (−12.1 kJ mol−1) of the mutation Phe14Asn arises from two factors: a reverse hydrophobic effect and the formation of a good N-cap at α-helix 1. In addition, a forward–backward hydrogen-bonding pattern, resembling an N-capping box and involving Asn14 and Arg18, has been found. The two Val to Thr mutations at the hydrophobic core have different thermodynamic effects: the mutation Val21Thr does not affect the stability of the protein while the mutation Val86Thr causes a small destabilization of 1.7 kJ mol−1. At site 21 a backward side-chain-to-backbone hydrogen bond is formed inside α-helix 1 with the carbonyl O atom of the i − 4 residue without movement of the mutated side chain. The destabilizing effect of introducing a polar group in the core is efficiently compensated for by the formation of an extra hydrogen bond. At site 86 the new Oγ atom escapes from the hydrophobic environment by a χ1 rotation into an adjacent hydrophilic cavity to form a new hydrogen bond. In this case the isosteric Val to Thr substitution is disruptive but the loss in stabilization energy is partly compensated by the formation of a hydrogen bond. The two crystal structures described in this work underline the significance of the hydrogen-bond component to protein stability.