Three-dimensional structures of the human alpha 2-macroglobulin-methylamine and chymotrypsin complexes.

The three-dimensional structures of chymotrypsin- and methylamine-treated negatively stained human alpha 2-macroglobulin have been determined by weighted back projection from electron microscope data. Projections of the reconstructions show good concordance with two-dimensional averages of both stained and frozen-hydrated molecules. The reconstructions reveal that the H-shaped front projection of the molecule is related to the smaller ellipsoidal end view by a rotation of 90 degrees about the crossbar (minor axis) of the H. This finding is in agreement with tilt studies. The reconstruction of the alpha 2-macroglobulin-methylamine reveals an hour-glass shaped void which is filled by the two proteinase molecules in the reconstruction of alpha 2-macroglobulin-chymotrypsin. Protein plugs which appear to block the exterior entrances to the cavity may function to prevent access of proteins to the encapsulated proteinase and serve to block its escape. Extensive thresholding of each reconstruction leaves a "backbone" consisting of two side-by-side rod-like structures, suggesting that this is the arrangement of the two protomeric units which form the molecule. Both structures show some departure from the expected symmetry. The asymmetries are robust features of the reconstructions and may reflect structurally asymmetric features of the transformation from the native to the chymotrypsin-treated form of the molecule.     

Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human α2-macroglobulin

α₂-Macroglobulin (α₂M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in α₂M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of α₂M-proteinase or α₂M-methylamine with α₂M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to α₂M was studied by electron microscopy. 7H11D6 bound to α₂M-methylamine and α₂M-trypsin but not to native α₂M. The structure of α₂M after conformational change resembled the letter “H.” 7H11D6 epitopes were identified near the apices of the four arms in the α₂M “H” structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to α₂M-trypsin but not to native α₂M). α₂M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete α₂M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary α₂M-trypsin (1 mole trypsin per mole α₂M instead of 2). Structures resembling the letter “H” were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated. These incompletely transformed structures were similar to the α₂M conformational intermediates described previously (S. L. Gonias and N. L. Figler (1989) J. Biol. Chem. 264, 9565–9570). We propose that lateral arm extension is a critical step in α₂M conformational change. Failure of lateral arm extension is probably a common property of different α₂M conformational intermediates.     

Domain 1 of Mucosal Addressin Cell Adhesion Molecule Has an I1-set Fold and a Flexible Integrin-binding Loop

Mucosal addressin cell adhesion molecule (MAdCAM) binds integrin α₄β₇. Their interaction directs lymphocyte homing to mucosa-associated lymphoid tissues. The interaction between the two immunoglobulin superfamily (IgSF) domains of MAdCAM and integrin α₄β₇ is unusual in its ability to mediate either rolling adhesion or firm adhesion of lymphocytes on vascular surfaces. We determined four crystal structures of the IgSF domains of MAdCAM to test for unusual structural features that might correlate with this functional diversity. Higher resolution 1.7- and 1.4-Å structures of the IgSF domains of MAdCAM in a previously described crystal lattice revealed two alternative conformations of the integrin-binding loop, which were deformed by large lattice contacts. New crystal forms in the presence of two different Fabs to MAdCAM demonstrate a shift in IgSF domain topology from the I2- to I1-set, with a switch of integrin-binding loop from CC′ to CD. The I1-set fold and CD loop appear biologically relevant. The different conformations seen in crystal structures suggest that the integrin-binding loop of MAdCAM is inherently flexible. This contrasts with rigidity of the corresponding loops in vascular cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, ICAM-2, ICAM-3, and ICAM-5 and may reflect a specialization of MAdCAM to mediate both rolling and firm adhesion by binding to different α₄β₇ integrin conformations.     

Histomorphometric analysis of adult articular calcified cartilage zone

The aim of this study was to carry out a histomorphometric analysis of calcified cartilage zone (CCZ) and its interfaces between hyaline cartilage and subchondral bone. The study used 40 donated normal human femoral condyles, from which paraffin-embedded sections were prepared after fixation and decalcification. The histomorphology of the CCZ were qualitatively and quantitatively observed by staining, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. The hyaline cartilage and CCZ were stained red with Safranin-O, and the subchondral bone was stained blue with Fast green. CCZ was stained black after von Kossa staining. The hyaline cartilage was interlocked tightly in the manner of “ravine-engomphosis” by the CCZ. The surface roughnesses of tidemark and cement line were 1.14 ± 0.04 and 1.99 ± 0.38. The maximum, minimum and mean thicknesses of CCZ were 277.12 ± 8.6, 9.83 ± 6.72 and 104.162 ± 0.87 μm, respectively. The cell density of CCZ (51.25 ± 21.26 cells/mm²) was significantly lower than that of the hyaline cartilage (152.54 ± 35.77 cells/mm²) (P < 0.05). The subchondral bone was anchored tightly in the manner of a “comb-anchor” by the CCZ in our 3D reconstruction model. Thus, we discovered two junctional interfaces of CCZ using different histomorphometric methods. The upper interface of CCZ is a “ravine-engomphosis” shape, while its lower interface is a “comb-anchor” shape.     

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)₄ complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)₂ lobes joined with D₂ symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)₄ complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)₄ kinase core, because a β₄ subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.     

Intramolecular disorder and its relation to mesophase structure in lipid/water mixtures

NMR spin-half pair dipolar echo measurements are reported for the lamellar (dispersions and multibilayer stacks) and hexagonal phases of potassium palmitate/²H₂O mixtures. In the lamellar L_β and L_γ (gel) phases the alkyl chains are rigid and perfectly ordered, while in the lamellar L_α and hexagonal phases they are flexible and disordered. In particular, the measurements show that in the fluid lamellar L_α phase the chain is “bent” at the C₉–C₁₀ segment; but is “straight” in the hexagonal phase.     

Karyophobic proteins : A category of abundant soluble proteins which accumulate in the cytoplasm

The cytoplasm of oocytes of Xenopus laevis is enriched in several soluble proteins which are either absent from the nucleus or are present there at very low concentrations. These molecules, collectively referred to as karyophobic (from the Greek verbs oβιν and oβλoθαi which are meant here in the sense of “to be afraid of” or “to avoid”) proteins represent more than 20% of the total soluble cytoplasmic proteins and include some of the most abundant soluble cellular components. They may be recovered from high-speed supernatant (S-100) fractions and, following sucrose gradient centrifugation, most of them appear in the form of complexes smaller than 8.5S. On denaturation in urea and two-dimensional gel electrophoresis these proteins appear to be comprised of polypeptides of widely different sizes (ca M_r 15 000–230 000) and isoelectric points covering a broad range of pH values (4.2–8.0). Gel filtration and isoelectric focusing of native karyophobic proteins show that the majority occur in acidic complexes smaller than M_r 150 000, including one case of a small karyophobic protein (C9; M_r 30 000). In contrast to karyophilic proteins and proteins equilibrating between nucleus and cytoplasm karyophobic soluble proteins from [³⁵S]methionine-labelled ooplasms, when injected into unlabelled oocytes, remain in the cytoplasm. Human proteins with a similar karyophobic behaviour have been identified in fractions of soluble proteins from HeLa cells; there, the major karyophobic protein (HCa, M_r 36 000) is also one of the most abundant soluble proteins.We conclude that the specific nucleocytoplasmic compartmentalization of soluble proteins is governed not only by the principles of exclusion of large molecules from nuclear uptake and the existence of karyophilic signals in certain proteins but that a series of soluble, globular proteins exist in the cytoplasm, which have other molecular features which selectively exclude them from distribution over the nucleus. The possible functional role of the selective enrichment of these abundant proteins, which so far have escaped attention, in establishing a cytoplasmic milieu is discussed.     

MR Image Reconstruction Using Block Matching and Adaptive Kernel Methods

An approach to Magnetic Resonance (MR) image reconstruction from undersampled data is proposed. Undersampling artifacts are removed using an iterative thresholding algorithm applied to nonlinearly transformed image block arrays. Each block array is transformed using kernel principal component analysis where the contribution of each image block to the transform depends in a nonlinear fashion on the distance to other image blocks. Elimination of undersampling artifacts is achieved by conventional principal component analysis in the nonlinear transform domain, projection onto the main components and back-mapping into the image domain. Iterative image reconstruction is performed by interleaving the proposed undersampling artifact removal step and gradient updates enforcing consistency with acquired k-space data. The algorithm is evaluated using retrospectively undersampled MR cardiac cine data and compared to k-t SPARSE-SENSE, block matching with spatial Fourier filtering and k-t ℓ₁-SPIRiT reconstruction. Evaluation of image quality and root-mean-squared-error (RMSE) reveal improved image reconstruction for up to 8-fold undersampled data with the proposed approach relative to k-t SPARSE-SENSE, block matching with spatial Fourier filtering and k-t ℓ₁-SPIRiT. In conclusion, block matching and kernel methods can be used for effective removal of undersampling artifacts in MR image reconstruction and outperform methods using standard compressed sensing and ℓ₁-regularized parallel imaging methods.     

Differential steric effects in the axial ligation of pyridine and imidazole to α- and β-alkylcobinamides

One subclass of B₁₂-requiring enzymes is now known to bind their B₁₂ coenzymes “base-off,” with a histidine residue from the protein supplying an imidazole ligand to the cobalt center. Recent results from Sirovatka and Finke (J.M. Sirovatka and R.G. Finke, J.Am. Chem. Soc. 119, (1997) 3057) show that imidazole has an extraordinary trans effect on the mode of carbon–cobalt bond cleavage in coenzyme B₁₂ analogs, compared to pyridine or the natural 5,6-dimethylbenzimidazole ligand, and it was suggested that a differential steric effect could, in part, account for the uniqueness of the imidazole ligand. Such a differential steric effect for imidazole and pyridine is now demonstrated by studies of the thermodynamics of ligation of these ligands to the α and β diastereomers of two alkylcobinamides (RCbi⁺s, derivatives of cobalamins which lack the normal axial nucleotide) based on the known differences in steric crowding of the α (“lower”) and β (“upper”) axial ligand positions of cobalt corrinoids. Imidazole binds more tightly than pyridine to both diastereomers of NCCH₂Cbi⁺ and CF₃Cbi⁺, in all cases due to a more favorable entropy change, which is the result of lowered steric interference with corrin side chain thermal motions.     

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

The L-type Ca²⁺ channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α_1s or Ca_V1.1, α₂, β_1a, δ1, and γ), we created transgenic mice expressing a recombinant β_1a subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α₂-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca_V1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α_1s subunit to the membrane and its suggested role in excitation-contraction coupling.     

Electron microscopic demonstration of a common structural motif in human complement factor C3 and rat α1 -inhibitor 3 (murinoglobulin)

Electron microscopy of two homologous giant proteins revealed that complement factor C3 and α_i-inhibitor 3 have a common structural motif of a semicircularly bent string 18–20 nm long with two or three bumps indicating globular domains. C3 had a structure similar to the letter C with a small but distinct hole in the center. α₁-Inhibitor 3 was a more complete ring sometimes ajar at one corner. When the latter was treated with a proteinase, it became slightly flattened and adopted a squarish C-shape.     

Prostaglandin release from the guinea-pig uterus superfused in vitro. Effect of stage of estrous cycle, progesterone, estradiol, oxytocin and A23187

Prostaglandin (PG) E₂ was the major PG released from the superfused guinea-pig uterus on Day 7, followed by in descending order 6-oxo-PGF_1α, thromboxane (TX) B₂ and PGF_2α. However, the outputs of all four substances were low and were very similar. By Day 15, PGF_2α output from the superfused uterus had increased 21.9-fold, whereas the outputs of PGE₂, 6-oxo-PGF_1α and TXB₂ had increased only 1.8-, 2.9- and 1.2-fold, respectively. A mechanism is apparently “switched on” between Days 7 and 15 which causes a fairly specific increase in the release of PGF_2α from the uterus.Progesterone and/or estradiol had no effect on PG or TX release when superfused over the uterus on Day 7, nor did they have any effect on PG and TX release from the Day 15 uterus when administered separately. When administered together, however, they significantly inhibited PGF_2α, PGE₂ and 6-oxo-PGF_1α, but not TXB₂, release from the Day 15 uterus. Oxytocin had no effect on PG release from the Day 7 or Day 15 uterus, while A23187 stimulated PGF_2α, 6-oxo-PGF_1α and, to a lesser extent, PGE₂ release from the uterus on both Days 7 and 15 Oxytocin is apparently not important for stimulating PGF_2α release from the guinea-pig uterus in relation to luteolysis, whereas increasing intracellular free Ca⁺⁺ levels may be part of the mechanism for “switching on” uterine PG synthesis. Furthermore, changes in intracellular free Ca⁺⁺ levels in the endometrium may be responsible for the pulsatile nature of PGF_2α release from the uterus.     

Crystal Structure of the Mg·ADP-inhibited State of the Yeast F1c10-ATP Synthase

The F₁c₁₀ subcomplex of the yeast F₁F₀-ATP synthase includes the membrane rotor part c₁₀-ring linked to a catalytic head, (αβ)₃, by a central stalk, γδϵ. The Saccharomyces cerevisiae yF₁c₁₀·ADP subcomplex was crystallized in the presence of Mg·ADP, dicyclohexylcarbodiimide (DCCD), and azide. The structure was solved by molecular replacement using a high resolution model of the yeast F₁ and a bacterial c-ring model with 10 copies of the c-subunit. The structure refined to 3.43-Å resolution displays new features compared with the original yF₁c₁₀ and with the yF₁ inhibited by adenylyl imidodiphosphate (AMP-PNP) (yF₁(I–III)). An ADP molecule was bound in both β_DP and β_TP catalytic sites. The α_DP-β_DP pair is slightly open and resembles the novel conformation identified in yF₁, whereas the α_TP-β_TP pair is very closed and resembles more a DP pair. yF₁c₁₀·ADP provides a model of a new Mg·ADP-inhibited state of the yeast F₁. As for the original yF₁ and yF₁c₁₀ structures, the foot of the central stalk is rotated by ∼40 ° with respect to bovine structures. The assembly of the F₁ central stalk with the F₀ c-ring rotor is mainly provided by electrostatic interactions. On the rotor ring, the essential cGlu⁵⁹ carboxylate group is surrounded by hydrophobic residues and is not involved in hydrogen bonding.     

The Novel Three-Dimensional Structure of Native Human α2-Macroglobulin and Comparisons with the Structure of the Methylamine Derivative

A three-dimensional reconstruction of α₂-macro-globulin (α₂M) was computed from stain images. The structure appears to have point group symmetry 222 and, as also revealed by a tilt experiment, has the gross shape of a oval that displays a ∼90° twist in the body of the molecule. The reconstruction reveals a novel structure that consists of two Z-shaped components arranged in opposite orientation. These shapes are interconnected by two bridges at the elbow bends of the Z and by two arch-like features that join their ends. The molecule has dimensions of ∼190 × 125 × 120 Å that encloses a 90° twisted ellipsoidal shaped central cavity of 70 × 35 Å. The cavity has four small openings arranged in a staggered configuration that extend to the outside. Serial slices of α₂M and α₂M-methylamine show that the bodies of the structures appear to be twisted in the opposite orientation. It is proposed that the four thioester bonds in the native molecule are responsible for maintaining its twisted configuration and that their cleavage with methylamine results in the structure becoming twisted in the opposite orientation. A comparison of average images derived from unstained particles of monoclonal Fab-labeled α₂M and α₂M-methylamine is consistent with this proposal. This unusual change in the handedness of α₂M may have an important role in the encapsulation of the proteinase.     

Recent advances in selective alpha1-adrenoreceptor antagonists as antihypertensive agents

Hypertension is one of the most serious health problems of the modern world with a continuous rise in the number of patients. Selective α₁-adrenoreceptor antagonists though have many advantages and uses in the management of arterial hypertension, their lack of specificity at the level of α₁-adr subtypes leads to multiple side effects. Existence of multiple α₁-adr subtypes holds great promise for the discovery and development of more specific and selective drug molecules, targeting only one α₁-adr subtype at a time and thus relative freedom from side effects. Herein, the research done on the discovery and evaluation of a variety of chemically diverse structures as selective antagonists of α₁-adr and α₁-adr subtypes in recent years has been reviewed.     

Integrin α5β1 Expression Is Required for Inhibition of Keratinocyte Migration by Ganglioside GT1b

Polysialoganglioside GT1b, a keratinocyte membrane glycosphingolipid, inhibits normal keratinocyte adhesion and migration on a fibronectin matrix. The specificity of the inhibition for cells plated on a fibronectin matrix and competition of GT1b inhibition with peptide RGDS suggest that GT1b abrogates the α₅β₁/fibronectin interaction. We examined the effects of GT1b on the adhesion and migration of keratinocyte-derived cell lines and correlated GT1b responsiveness and α₅β₁integrin expression. GT1b (5 nM) significantly inhibited migration of normal human keratinocytes, immortalized keratinocytes, and squamous cell carcinoma SCC12F2 cells on fibronectin, but not on collagen I. Concentrations as high as 5 μM had no effect on SCC13 or HaCaT cells. Likewise, GT1b inhibited fibronectin-dependent cell adhesion of normal human keratinocytes, immortalized keratinocytes, and SCC12F2 cells, but had no effect on SCC13 or HaCaT cells. Flow cytometric and Western immunoblot analysis of integrin expression showed significantly decreased α₅and β₁integrin expression in SCC13 and HaCaT cells compared to normal keratinocytes, immortalized keratinocytes, and SCC12F2 cells. Incubation with TGF-β1 increased α₅β₁integrin expression and induced responsiveness to GT1b in HaCaT cells. These data imply that GT1b “response” requires sufficient expression of α₅β₁and further suggest that the mechanism of the inhibitory effect of GT1b involves GT1b/α₅β₁interaction.     

Localization of β1-Adrenergic Receptors in the Cochlea and the Vestibular Labyrinth

Sympathetic activation in a “fight or flight reaction” may put the sensory systems for hearing and balance into a state of heightened alert via β₁-adrenergic receptors (β₁-AR). The aim of the present study was to localize β₁-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize β₁-AR by Western immunoblots, and to identify β₁-AR pharmacologically by measurements of cAMP production. Staining for β₁-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated β₁-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, β₁-AR are present and functional in inner ear epithelial cells that are involved in K⁺ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.     

Direct and Regulated Interaction of Integrin αEβ7 with E-Cadherin

The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)–1, vascular cell adhesion molecule (VCAM)–1, or mucosal addressin cell adhesion molecule (MadCAM)–1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, α_Eβ₇, has been proposed. Here, we demonstrate that a human E-cadherin–Fc fusion protein binds directly to soluble recombinant α_Eβ₇, and to α_Eβ₇ solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY′ cells expressing the α_Eβ₇ integrin adhere strongly to purified E-cadherin–Fc coated on plastic, and the adhesion can be inhibited by antibodies to α_Eβ₇ or E-cadherin.

The binding of α_Eβ₇ integrin to cadherins is selective since cell adhesion to P-cadherin–Fc through α_Eβ₇ requires >100-fold more fusion protein than to E-cadherin–Fc. Although the structure of the α_E-chain is unique among integrins, the avidity of α_Eβ₇ for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of α_Eβ₇ for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the α_Eβ₇ integrin.

     

The crystal and molecular structure of 1,6-anhydro-β-d-mannopyranose

The crystal structure of 1,6-anhydro-β-d-mannopyranose, C₆H₁₀O₅, is orthorhombic, P2₁2₁2₁, with a = 10.971(2), b = 13.935(3), c = 9.012(1) Å, V = 1377.76 »³ (MoKα, λ = 0.7107 Å), Z = 8, D_x = 1.563 M.gm⁻³, D_m = 1.565 M.gm⁻³. the structure was solved by MULTAN and refined to R(F) = 0.043 for 2355 reflections. The two symmetry-independent molecules in the unit cell have similar conformations, except for the orientation of one of the three hydroxyl groups. The conformation of the pyranose rings is ¹C₄ distorted towards E_o, and that of the anhydro rings is E. There are significant differences between the two molecules in two of the four C---O bond-lengths. These occur where there are important differences in the hydrogen-bonding environment of the oxygen atoms. The molecules are hydrogen-bonded by three linear and three bifurcated O---H···O interactions which form four-membered loops linked into infinite chains. Empirical force-field calculations with MMI-CARB reproduced the geometry of the molecules within the variations observed experimentally between the two molecules, except for a C---O bond in one of the molecules. The effect of excluding the anomeric effect from the theoretical calculations was not significant. Calculations for an intramolecularly hydrogen-bonded molecule were also carried out as a model for the molecules in a non-polar solvent.