Cryoelectron-Microscopy Image Reconstruction of Symmetry Mismatches in Bacteriophage φ29

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The Podoviridae phage C1 was one of the earliest isolated bacteriophages and the first virus documented to be active against streptococci. The icosahedral and asymmetric reconstructions of the virus were calculated using cryo-electron microscopy. The capsid protein has an HK97 fold arranged into a T ¼ 4 icosahedral lattice. The C1 tail is terminated with a φ29-like knob, surrounded by a skirt of twelve long appendages with novel morphology. Several C1 structural proteins have been identified, including a candidate for an appendage. The crystal structure of the knob has an N-terminal domain with a fold observed previously in tube forming proteins of Siphoviridae and Myoviridae phages. The structure of C1 suggests the mechanisms by which the virus digests the cell wall and ejects its genome. Although there is little sequence similarity to other phages, conservation of the structural proteins demonstrates a common origin of the head and tail, but more recent evolution of the appendages.bacteriophage C1 tail | tail knob | X-ray crystallography

Section: Resultsmentioning

confidence: 99%

Structural investigations of a Podoviridae streptococcus phage C1, implications for the mechanism of viral entry

Aksyuk

Bowman

Kaufmann

et al. 2012

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“…Electron micrographs were recorded at a magnification of 33,000 with a CM300 FEG microscope under low-dose conditions. Both mutant virus reconstructions assumed no symmetry and were based on a procedure as described (4,7,34). The N-and C-terminal domains of gp13 were initially fitted manually into the cryoEM density map by using the program Chimera and then optimized with the program EMfit, assuming 2-fold symmetry.…”

Section: Methodsmentioning

confidence: 99%

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage φ29 tail

Xiang

Morais

Cohen

et al. 2008

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The small bacteriophage 29 must penetrate the Ϸ250-Å thick external peptidoglycan cell wall and cell membrane of the Grampositive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage 29 is noncontractile and Ϸ380 Å long. A 1.8-Å resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N-and C-terminal domains, whose structures resemble lysozyme-like enzymes and metalloendopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal end of the 29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13 ؊ mutants with the 29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.hydrolase ͉ infection ͉ structure ͉ zinc ion

“…S3). In addition, a hexameric pRNA ring would presumably have six, rather than five, projecting spokes in cryo-EM recon- struction (8). Enforcing sixfold symmetry during reconstruction (8,26) yielded a ring, but without the spokes known to be comprised of P A (3) (approximately 45% of the 120b pRNA mass) due to the imposition of sixfold averaging.…”

Section: Two Flexible Joints and Lack Of Tertiary Interactions In Thementioning

confidence: 99%

“…The requirement of a RNA-ring structure in the assembly and function of the ϕ29 DNA packaging motor contrasts with equivalent motors in other dsDNA phages such as T4, SPP1, lambda, and P22 where protein-protein contacts anchor ring ATPases to the phage proheads (1). Cryoelectron microscopy (cryo-EM) 3D reconstructions (2,3,8) show that pRNA is strategically positioned in the ϕ29 packaging motor to link the capsid, connector and ATPase components of the motor (Fig. 1A).…”

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confidence: 99%

Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

Ding

Wang

et al. 2011

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138

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage ϕ29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular basepairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of ϕ29 DNA.D uring the assembly of the Bacillus subtilis bacteriophage ϕ29 and other double-stranded DNA phages, an ATP-driven ring motor plays a crucial role in packaging viral DNA to near crystalline density inside preformed protein shells (proheads) (1). In ϕ29, the packaging motor is comprised of three ring structures (Fig. 1A). The head-tail connector, a dodecameric ring of gp10, is embedded in the portal vertex of the head and provides a channel for entry and exit of the genome (2). An oligomeric ring of ϕ29 encoded prohead RNA (pRNA) encircles the protruding end of the connector, displaying five "spokes" that project away from the head (2, 3). Subunits of the viral packaging ATPase gp16, a member of the FtsK/HerA ring motor family (4), form a ring that contacts the five spokes of pRNA, completing the packaging motor (3, 5, 6). Single-molecule laser tweezers studies showed that the ϕ29 DNA packaging motor is one of the strongest molecular motors known, generating approximately 65 pN force (compared to approximately 3 pN for muscle myosin) (7). The requirement of a RNA-ring structure in the assembly and function of the ϕ29 DNA packaging motor contrasts with equivalent motors in other dsDNA phages such as T4, SPP1, lambda, and P22 where protein-protein contacts anchor ring ATPases to the phage proheads (1). Cryoelectron microscopy (cryo-EM) 3D reconstructions (2,3,8) show that pRNA is strategically positioned in the ϕ29 packaging motor to link the capsid, connector and ATPase components of the motor (Fig. 1A).Besides being an essential component of the motor, the ϕ29 pRNA multimer is a rare example of a self-assembling RNA that functions at the quaternary...