Mohammed O. Badasso

Bacteriophage φ29 scaffolding protein gp7 before and after prohead assembly

Three-dimensional structures of the double-stranded DNA bacteriophage φ29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 Å, respectively. Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail. The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis. DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release. A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells. The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers.

Structure determination of the head–tail connector of bacteriophage φ29

The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map. The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

Crystallization and X-ray analysis of the Y75N mutant of Mucor pusillus pepsin complexed with inhibitor.

Y75N mutant Mucor pusillus pepsin has been overexpressed in yeast, purified and cocrystallized with the iodine-containing human renin inhibitor CP-113972 [(2R,3S]-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S-methyl-cysteinyl)amino-4]-cyclohexyl-2-hydroxybutanoate] for X-ray crystallography. Tetragonal complex crystals with space group P4(3)2(1)2 were produced by the hanging-drop vapour-diffusion method and diffracted to 3.0 A. The crystals exhibited unit-cell parameters a = b = 182.5, c = 99.1 A and contained four molecules in the asymmetric unit. A 96% complete data set was collected at 298 K using Cu Kalpha X-rays from a rotating-anode generator. Solution of the crystal structure of Y75N mutant M. pusillus pepsin is under way by molecular replacement using the molecular coordinates of wild-type M. pusillus pepsin as a model.

Erratum: Detailed architecture of a DNA translocating machine: The high-resolution structure of the bacteriophage φ29 connector particle (Journal of Molecular Biology (2002))

The recent paper by Guasch et al.1 describing the structure of the ϕ29 bacteriophage head–tail connector at 2.1 A resolution (PDB code 1h5w), makes reference to the original 3.2 A resolution structure (PDB code 1fou) published by Simpson et al.2 and used by Guasch et al. to solve the 1h5w structure by molecular replacement. The Guasch paper states that there are marked differences between the 1h5w and 1fou structures in the chain tracing and the side-chain positioning in the wide domain. As these and a number of other statements in the Guasch paper may have led to the impression that these differences are larger than they really are, we give here Tables summarizing the similarities and differences (Tables 1 and ​and22). Table 1 Statistical comparison of the ϕ29 connector wide domains in different structures Table 2 Superposition of the entire monomers in the four reported structures Another paper by Simpson et al.,3 which was accidentally omitted from the text of the Guasch et al. paper, reports two additional structures of the ϕ29 connector: at 3.2 A resolution and at 2.9 A resolution (PDB codes 1jnb and 1ijg, respectively). The 2.9 A resolution structure shows no significant differences from the structure subsequently reported by Guasch et al. (Figure 1; Tables 1 and ​and22). Figure 1 Stereo diagram showing the superposition of the Cα back-bone of monomer A in the 2.1 A resolution structure 1h5w (green) and the 2.9 A resolution 1ijg structure (red).

Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage ϕ29

The Bacillus subtilis bacteriophage phi29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 77.13, c = 37.12 A. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 A. Complete data sets have been collected to 1.78 and 1.80 A for forms I and II, respectively, at 100 K using Cu Kalpha X-rays from a rotating-anode generator. Calculation of a VM value of 2.46 A3 Da(-1) for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a VM of 4.80 A3 Da(-1) with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.

Structure of the bacteriophage ϕ29 DNA packaging motor

In a digital communication system in which the information is transmitted by successions of bits termed packets, stations may be called upon to perform the function of a relay between other stations. The stations which may be called upon to perform the function of a relay comprise: a checking device which merely checks the address code of a packet received; an eliminating and switching device which either destroys the packet (doubtful address code) or orients the packet toward receiving means pertaining to the station (address code identical to the code of the station) or toward the station corresponding to the address code by passing through a transit memory; and information producing means for producing packets within the station. The contents of the transit memory are transmitted in priority by the station. The packets produced within a station are transmitted in the gaps between the packets coming from the transit memory and in an order which is a function of a classification between the priorities allocated to the information producing means of the considered station.

Purification, co-crystallization and preliminary X--ray analysis of the natural aspartic proteinase inhibitor IA3 complexed with saccharopepsin from Saccharomyces cerevisiae.

The vacuolar aspartic proteinase from bakers yeast, saccharopepsin, has been co-crystallized with its natural inhibitor I(A)3, found in the cytosol. The I(A)3-saccharopepsin complex crystals belong to the space group P6(2)22, with unit-cell parameters a = b = 192.1, c = 59. 80 A and one molecule per asymmetric unit. The initial X-ray analysis of the complex indicates that the crystals diffract to 5.0 A, similar to native saccharopepsin crystals. This is probably a consequence in part of glycosylation of the native saccharopepsin. Full structural analysis of the complex crystal is in progress.