The projection structure of EmrE, a proton-linked multidrug transporter from Escherichia coli, at 7 A resolution

EmrE belongs to a family of eubacterial multidrug transporters that confer resistance to a wide variety of toxins by coupling the influx of protons to toxin extrusion. EmrE was purified and crystallized in two dimensions by reconstitution with dimyristoylphosphatidylcholine into lipid bilayers. Images of frozen hydrated crystals were collected by cryo-electron microscopy and a projection structure of EmrE was calculated to 7 A resolution. The projection map shows an asymmetric EmrE dimer with overall dimensions approximately 31 x 40 A, comprising an arc of highly tilted helices separating two helices nearly perpendicular to the membrane from another two helices, one tilted and the other nearly perpendicular. There is no obvious 2-fold symmetry axis perpendicular to the membrane within the dimer, suggesting that the monomers may have different structures in the functional unit.     

Crystallization and preliminary X-ray analysis of pig kidney DOPA decarboxylase.

DOPA decarboxylase from pig kidney, an alpha 2 dimeric enzyme of Mr = 107,000, has been crystallized by the vapour diffusion method with ammonium sulphate as precipitant. The crystals belong to the space group P6(2) (or its enantiomer P6(4)) and have unit cell dimensions of a = b = 155.9 A, c = 87.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. They diffract to 2.6 A resolution. There is one dimeric molecule per asymmetric unit. Rotation function studies have revealed the orientation of the non-crystallographic 2-fold axis of the dimer in the asymmetric unit.     

Structure of the cytochrome c oxidase dimer electron microscopy of two-dimensional crystals

We have studied the structure of beef heart mitochondrial cytochrome c oxidase dimers by image-processing of electron micrographs of the vesicle crystal form. Specimens were prepared by different procedures, which contrast different features of the crystals. Heavy-atom shadowing of freeze-dried crystals contrasts the exterior or M-side surface (mitochondrial matrix-side) and reveals a 100 Å long ellipsoidal dimer oriented with its long axis in the (?1, 1) direction of the 95 Å × 125 Å rectangular unit cell. The M-side surface structure correlates well with the intra-bilayer structure revealed by contrast matching extra-bilayer protein with glucose. Frozen suspensions of vesicle crystals fracture predominantly along hydrophilic surfaces revealing the interior C-side (mitochondrial cytoplasm-facing surface) of vesicle crystals. The C-side surface revealed in shadowed replicas of fracture surfaces shows the ends of the dimers furthest from the bilayer surface; they consist of two structural domains separated by 70 to 80 Å. We present a new interpretation of the structure of the cytochrome oxidase dimer based on these data and on the y-shaped monomer structure described by Fuller et al. (1979). A cytochrome oxidase dimer is formed from two y-shaped monomers joined along one set of identical M-domain arms with the other arms approximately 70 Å apart along a unit cell diagonal in the (?1, 1) direction. The arms of the monomers lie within and perpendicular to the phospholipid bilayer, and they protrude approximately 25 Å beyond the bilayer surface on the M-side. The y tails represent the C-side domains, which are closely apposed across the dimer 2-fold axis near the C-side bilayer surface. Further away from the bilayer surface, C-side domains split away from one another forming a large cleft.     

Packing analysis of crystalline myosin subfragment-1. Implications for the size and shape of the myosin head

Crystals of myosin subfragment-1 have been examined by X-ray diffraction and electron microscopy to determine how the molecules pack in the unit cell and to gain preliminary information on the size and shape of the myosin head. Subfragment-1 crystallizes in space group P212121. Analysis of the X-ray diffraction photographs shows that there are eight molecules in the unit cell with two in the asymmetric unit related by a non-crystallographic or local 2-fold axis. It also indicates that in projection down the a axis, two molecules of myosin subfragment-1 lie almost directly on top of one another except for a translation of about 9 A along c. Small crystals were fixed and embedded in the presence of tannic acid, and thin sections were cut perpendicular to each of the three crystallographic axes. Image analysis of micrographs recorded from these sections confirm the packing arrangement deduced from X-ray diffraction, and give the approximate size and shape of the molecule in the crystal lattice. They show that the molecule is at least 160 A long with a maximum thickness of about 60 A, and that it has marked curvature in the unit cell.     

Complementation of RNA binding site mutations in MS2 coat protein heterodimers.

The coat protein of bacteriophage MS2 functions as a symmetric dimer to bind an asymmetric RNA hairpin. This implies the existence of two equivalent RNA binding sites related to one another by a 2-fold symmetry axis. In this view the symmetric binding site defined by mutations conferring the repressor-defective phenotype is a composite picture of these two asymmetric sites. In order to determine whether the RNA ligand interacts with amino acid residues on both subunits of the dimer and in the hope of constructing a functional map of the RNA binding site, we performed heterodimer complementation experiments. Taking advantage of the physical proximity of their N- and C-termini, the two subunits of the dimer were genetically fused, producing a duplicated coat protein which folds normally and allows the construction of the functional equivalent of obligatory heterodimers containing all possible pairwise combinations of the repressor-defective mutations. The restoration of repressor function in certain heterodimers shows that a single RNA molecule interacts with both subunits of the dimer and allows the construction of a functional map of the binding site.     

Cytochrome oxidase: structural insights from electron microscopy and from secondary structure prediction

Electron microscopic images of selectively contrasted cytochrome oxidase dimer crystals are interpreted in a manner consistent with the structure of monomers determined by Fuller et al. (J. Molec. Biol. 134, 305-327). The arms of the y-shaped monomers lie within and perpendicular to the lipid bilayer protruding approximately 25 A on the matrix side of the membrane. The cytoplasmic-side tails of two monomers spread apart in a dimer forming a large cleft. Decoration of the exposed matrix side of vesicle crystals with antisubunit IV antibody fragments indicates that subunit IV lies along the a-crystal axis roughly 20 A from the center of the dimer. A membrane propensity algorithm applied to the sequences of cytochrome oxidase subunits predicts a total of 19 transmembrane alpha-helices per monomer.     

Three-dimensional structure of ubiquinol: Cytochrome C reductase from Neurospora mitochondria determined by electron microscopy of membrane crystals

Membrane crystals have been prepared from mitochondrial ubiquinol: cytochrome c reductase by mixing the enzyme-Triton complex with phospholipid-Triton micelles and subsequently removing the Triton. The electron micrographs of the negatively stained crystals diffract to 2·5 nm, with unit cell dimensions of 13·7 nm by 17·4 nm. The enzyme is arranged in a two-sided plane group P22₁2₁, i.e. alternate molecules span the bilayer in an up and down manner. By combining tilted views of the membrane crystals, a low-resolution three-dimensional structure of the enzyme has been calculated. The structure shows that the enzyme is a dimer, the monomers being related by a 2-fold axis running perpendicular to the membrane. The monomeric units of the enzyme are elongated, extending approximately 15 nm across the membrane. The protein is unequally distributed with about 30% of the total mass located in the bilayer, 50% in a section which extends 7 nm from one side of the bilayer and 20% in a section which extends 3 nm from the opposite side of the bilayer. The two monomeric units are in contact only in the membraneous section. This structure is compared with a model of the enzyme which is derived from biochemical properties of the isolated subunits.     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.     

Preliminary crystallographic data for transketolase from yeast

Crystals of the vitamin B1-dependent enzyme transketolase from baker's yeast have been grown from the apo- and the holoform of the enzyme, using PEG as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell constants a = 76.3 A, b = 114.2 A, and c = 163.5 A. The crystals are stable in the x-ray beam and diffract to at least 2.2 A on a conventional x-ray source. The enzyme is a dimer of identical subunits, and a Vm value of 2.2 A/dalton indicates that the asymmetric unit contains a dimer. Rotation function calculations using native data (10-5 A) revealed a local 2-fold rotation axis with phi = 0 degree and omega = 20 degrees.     

Perfect hemihedral twinning in crystals of the γ-subunit of translation initiation factor 2 from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>: Cause and effect

The crystal structure of the γ-subunit of translation initiation factor 2 from the archaeon Sulfolobus solfataricus (SsoIF2γ) has been solved based on perfectly hemihedral twinned data. The protein was cocrystallized with the 10-fold molar excess of GTP analog (GDPCP) over protein. However, no nucleotide was found in the structure, and the model demonstrated the apo form of the protein. Two slightly different molecules in the asymmetric unit of the crystal are related by the non-crystallographic 2-fold axis and form a tightly associated dimer. This dimer is stabilized by an intermolecular hydrophobic core and hydrogen bonds. Lack of GDPCP in the nucleotide-binding pocket of the γ-subunit and significant excess of dimers over monomers in the crystallization solution suggest that these dimers are the building blocks of the crystal. Contrary to SsoIF2γ monomers, these dimers are able to crystallize in two oppositely oriented slightly different crystal domains, thus forming a twinned crystal. Comparison of crystallization conditions for the twinned and untwinned crystals of apo SsoIF2γ showed that stabilization of the dimers in the solution may be caused by higher sodium salt concentration. Since amino acid residues involved in intermolecular contacts in the dimer are responsible for binding of the γand α-subunits within SsoIF2, increase in sodium salt concentration may prevent functioning of SsoIF2 in the cell.     

Domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding

The crystal structure of griffithsin, an antiviral lectin from the red alga Griffithsia sp., was solved and refined at 1.3 A resolution for the free protein and 0.94 A for a complex with mannose. Griffithsin molecules form a domain-swapped dimer, in which two beta strands of one molecule complete a beta prism consisting of three four-stranded sheets, with an approximate 3-fold axis, of another molecule. The structure of each monomer bears close resemblance to jacalin-related lectins, but its dimeric structure is unique. The structures of complexes of griffithsin with mannose and N-acetylglucosamine defined the locations of three almost identical carbohydrate binding sites on each monomer. We have also shown that griffithsin is a potent inhibitor of the coronavirus responsible for severe acute respiratory syndrome (SARS). Antiviral potency of griffithsin is likely due to the presence of multiple, similar sugar binding sites that provide redundant attachment points for complex carbohydrate molecules present on viral envelopes.     

Control of crystal forms of apoferritin by site-directed mutagenesis

Surface charges of protein molecules are not only important to biological functions but also crucial to the molecular assembly responsible for crystallization. Appropriate alteration in the surface charge distribution of a protein molecule induces new molecular alignment in the proper direction in the crystal and, hence, controls the crystal form. Apoferritin molecules are known to crystallize in two- and three-dimensional forms in the presence of cadmium ions, which bridge neighboring protein molecules. Here we report a controlled transformation of the apoferritin 2-D crystal by site-directed mutagenesis. In mutant apoferritin, two amino acid residues binding a cadmium-ion through their negative charge, were replaced by one type of nonionic amino acid residues. The amino acid residues, Asp-84 and Gln-86 in the sequence of recombinant (i.e., wild-type) horse L -apoferritin, were replaced by Ser. The wild-type apoferritin yielded a hexagonal lattice 2-D crystal in the presence of cadmium ions. In contrast, the mutant apoferritin yielded two types of oblique crystals independent of the presence of cadmium ions. Image reconstruction of electron micrographs of the mutant crystals made clear that the mutant apoferritin molecules oriented themselves with the 2-fold symmetry axis perpendicular to the crystal plane in both crystals, while the wild-type apoferritin molecules oriented themselves with the 3-fold symmetry axis perpendicular to the crystal plane. The changes of crystal forms and molecular orientation in the 2-D crystals were well explained by a change of the electrostatic interactions induced by the mutagenesis. © 1995 Wiley-Liss, Inc.     

Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum

The three-dimensional structure of scallop sarcoplasmic reticulum membranes has been determined from electron micrographs of two classes of stain-filled tubules by helical reconstruction methods. These structures are characterized by dimer ribbons of Ca2+-ATPase molecules running diagonally around the tube wall. Deep right-handed grooves separate the ribbons. The elongated, curved units of the dimer (approximately 95 A long in the radial direction; 60 to 70 A axially, and about 30 A wide) are displaced axially by approximately 34 A and are connected at their outer ends by a bridge running nearly parallel to the tube axis. The monomers make a second contact at their inner ends. Adjacent units with the same orientation form a strong contact that is responsible for the ribbon appearance. Comparison of tubules of different diameter shows that one set of connections between the dimer ribbons is conserved: the inner ends of axially displaced dimers appear to make contact along a left-handed path almost perpendicular to the major grooves. The lipid bilayer cannot be clearly identified. The two-dimensional map obtained from flattened tubules is consistent with the three-dimensional reconstruction in showing dimer ribbons connected by a weak contact across the grooves, strongly resembling the inter-dimer bond observed in three dimensions. The two-dimensional map shows a 2-fold axis relating units of the dimer, but the three-dimensional tubes show a slight axial polarity that may arise from the presence of proteins other than the Ca2+-ATPase.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

Probing the structure of the dimeric KtrB membrane protein

The KtrAB ion transporter is a complex of two proteins, KtrB and KtrA. The integral membrane protein KtrB is expected to adopt the structural architecture typified by the pore domain of potassium channels. Here we show that homo-dimerization of KtrB proteins is most likely a general property of this family of transporters. Using cysteine mutants and bifunctional cross-linkers we define regions of the Bacillus subtilis KtrB molecule that are close to the molecular 2-fold axis and to the dimer interface. Fitting of the cross-linking data to a potassium channel-like model suggests structural similarities between potassium channels and KtrB proteins in the extracellular half of the molecule and differences in the cytoplasmic regions.     

Crystallization and preliminary X-ray analysis of porcine synovial collagenase

Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group I4, with unit cell dimensions a = b = 160.0 A, c = 53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4 A.     

Three-dimensional structure of ferricytochrome c' from Rhodospirillum rubrum at 2.8 A resolution.

The structure of ferricytochrome c' extracted from Rhodospirillum rubrum has been determined by the X-ray crystallographic method. Crystals in hexagonal space group P6(1), with unit-cell dimensions a = b = 51.72 A and c = 155.49 A, contain one dimer molecule composed of chemically identical polypeptide chains (monomer I and monomer II) per asymmetric unit. An electron density map has been calculated at a resolution of 2.8 A by the multiple isomorphous replacement method using four-circle diffractometer data from native crystals and two heavy-atom derivatives. The quality of the map was improved by averaging the electron density about the non-crystallographic 2-fold axis relating the two monomers. The initial three-dimensional model of monomer I was built on a computer graphics system and that of monomer II was derived from monomer I using the non-crystallographic symmetry matrices. The dimer structure has been refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The current model includes 244 amino acid residues (122 x 2) and 2 hemes, with a root-mean-square deviation in bond lengths from ideal values of 0.022 A. The current crystallographic R-factor is 23.3% for 4,481 independent reflections [magnitude of Fo greater than or equal to sigma (F)] between 5.0 and 2.8 A resolution. The monomer molecule is structurally organized as an array of four nearly parallel alpha-helices which construct a left-twisted bundle. One end of the bundle, in which a covalently bound protoheme IX prosthetic group is incorporated, is more divergent than the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary crystallographic study of glycosylated recombinant human renin

Single crystals of glycosylated recombinant human renin have been obtained using the hanging-drop vapor diffusion method with polyethylene glycol and sodium chloride as coprecipitants. The crystals belong to the cubic space group P2(1)3 with a = 143.0 A and contain two molecules of renin in the asymmetric unit. A self-rotation function study using 5.5 A data shows the orientation of a non-crystallographic 2-fold axis relating these two monomers.     

X-ray structure of lipoamide dehydrogenase from Azotobacter vinelandii determined by a combination of molecular and isomorphous replacement techniques

The crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii has been determined by a combination of molecular replacement and isomorphous replacement techniques yielding eventually a good-quality 2.8 A electron density map. Initially, the structure determination was attempted by molecular replacement procedures alone using a model of human glutathione reductase, which has 26% sequence identity with this bacterial dehydrogenase. The rotation function yielded the correct orientation of the model structure both when the glutathione reductase dimer and monomer were used as starting model. The translation function could not be solved, however. Consequently, data for two heavy-atom derivatives were collected using the Hamburg synchotron facilities. The derivatives had several sites in common, which was presumably a major reason why the electron density map obtained by isomorphous information alone was of poor quality. Application of solvent flattening procedures cleaned up the map considerably, however, showing clearly the outline of the lipoamide dehydrogenase dimer, which has a molecular weight of 100,000. Application of the "phased translation function", which combines the phase information of both isomorphous and molecular replacement, led to an unambiguous determination of the position of the model structure in the lipoamide dehydrogenase unit cell. The non-crystallographic 2-fold axis of the dimer was optimized by several cycles of constrained-restrained least-squares refinement and subsequently used for phase improvement by 2-fold density averaging. After ten cycles at 3.5 A, the resolution was gradually extended to 2.8 A in another 140 cycles. The 2.8 A electron density distribution obtained in this manner was of much improved quality and allowed building of an atomic model of A. vinelandii lipoamide dehydrogenase. It appears that in the orthorhombic crystals used each dimer is involved in contacts with eight surrounding dimers, leaving unexplained why the crystals are rather fragile. Contacts between subunits within one dimer, which are quite extensive, can be divided into two regions separated by a cavity. In one of the contact regions, the level of sequence identity with glutathione reductase is very low but it is quite high in the other. The folding of the polypeptide chain in each subunit is quite similar to that of glutathione reductase, as is the extended conformation of the co-enzyme FAD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystallization and preliminary crystallographic study of an octa-heme cytochrome c3 from Desulfovibrio desulfuricans Norway.

An octa-heme cytochrome c3, isolated as a dimeric molecule of about 30 kDa from the anaerobic bacteria Desulfovibro desulfuricans Norway, has been crystallized in a form suitable for atomic resolution X-ray structural investigations. The crystals are trigonal, space group P3(1)21 (or its enantiomorph P3(2)21), with cell dimensions: a = b = 72.9 A c = 62.7 A. The asymmetric unit contains most probably one monomer and a solvent content of about 60%. Under this assumption, the crystallographic 2-fold axis relates the two subunits of the dimer. Diffraction extends to 2.0 A.