Cryo-transmission electron tomography of native casein micelles from bovine milk.

Caseins are the principal protein components in milk and an important ingredient in the food industry. In liquid milk, caseins are found as micelles of casein proteins and colloidal calcium nanoclusters. Casein micelles were isolated from raw skim milk by size exclusion chromatography and suspended in milk protein-free serum produced by ultrafiltration (molecular weight cut-off of 3 kDa) of raw skim milk. The micelles were imaged by cryo-electron microscopy and subjected to tomographic reconstruction methods to visualize the 3-dimensional and internal organization of native casein micelles. This provided new insights into the internal architecture of the casein micelle that had not been apparent from prior cryo-transmission electron microscopy studies. This analysis demonstrated the presence of water-filled cavities (∼20 to 30 nm in diameter), channels (diameter greater than ∼5 nm), and several hundred high-density nanoclusters (6 to 12 nm in diameter) within the interior of the micelles. No spherical protein submicellar structures were observed.

In vitro assembly of bacteriophage P4 procapsids from purified capsid and scaffolding proteins.

Bacteriophage P4 is a satellite virus of bacteriophage P2, which has acquired the ability to utilize the structural gene products of P2 to assemble its own capsid. The normal P2 capsid has a T = 7 icosahedral structure comprised of the gpN-derived capsid protein, whereas the capsid produced under the control of P4 has a smaller, T = 4 structure. The protein responsible for this size determination is the P4-coded gene product Sid, which forms an external scaffold on the P4 procapsid. Using an in vitro assembly system, we show that gpN and Sid can coassemble into procapsid-like particles, indistinguishable from those produced in vivo, in the absence of any other gene products. The fidelity of the assembly reaction is enhanced by the inclusion of PEG and has a pH optimum between 8.0 and 8.5. Analysis of the assembly properties of truncated versions of Sid and gpN suggests that the amino-terminal part of Sid is involved in gpN binding, while the carboxyl-terminal part forms trimeric Sid-Sid interactions, and that the first 31 amino acids of gpN are required for binding to Sid as well as for size determination.

Freedom and restraint: themes in virus capsid assembly.

Viruses assemble protective capsids from several copies of one or a few structural proteins. This is accomplished through a combination of conformational flexibility and control mechanisms that restrict this flexibility. This review will discuss some of these mechanisms in light of the many recent results in this area.

X-ray crystallographic structure of the Norwalk virus capsid.

Norwalk virus, a noncultivatable human calicivirus, is the major cause of epidemic gastroenteritis in humans. The first x-ray structure of a calicivirus capsid, which consists of 180 copies of a single protein, has been determined by phase extension from a low-resolution electron microscopy structure. The capsid protein has a protruding (P) domain connected by a flexible hinge to a shell (S) domain that has a classical eight-stranded beta-sandwich motif. The structure of the P domain is unlike that of any other viral protein with a subdomain exhibiting a fold similar to that of the second domain in the eukaryotic translation elongation factor-Tu. This subdomain, located at the exterior of the capsid, has the largest sequence variation among Norwalk-like human caliciviruses and is likely to contain the determinants of strain specificity and cell binding.

The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus phiX174.

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174. Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.

Scaffolding proteins and their role in viral assembly.

Scaffolding proteins are proteins that are required to catalyse, regulate or modulate some step in the assembly of a macromolecular complex. They associate specifically with the nascent protein complex during assembly, but are subsequently removed, and are absent from the mature structure. Scaffolding proteins have been described primarily from viral systems, in particular from the double-stranded DNA bacteriophages, but most likely play a more general role in macromolecular assembly, a fundamental process in all biological systems. Scaffolding proteins may act in a specific fashion, by actively encouraging the formation of correct protein-protein interactions, or more generally by nucleating and promoting assembly. They may also work to ensure the fidelity of the assembly process by preventing the formation of improper interactions, in many ways similar to the role of molecular chaperones in protein folding. In viruses, scaffolding proteins are found both in the form of internal cores and external bracing, and may form elaborate and complex structures. This review will focus on the viral scaffolding proteins, for which an increasing amount of structural and functional information has recently become available.

Structure determination of the phiX174 closed procapsid.

The structure of a procapsid of the single-stranded DNA bacteriophage ++phiX174 was determined to 3.5 A resolution. The crystal space group was I213 with a unit-cell length of 774 A. The unit cell contained 16 icosahedral virus particles, each situated on a crystallographic threefold axis. Thus, there are two independent one-thirds of a particle per asymmetric unit, and a total of 40-fold non-crystallographic redundancy. To aid in the interpretation of the packing arrangement, crystals were prepared for thin sectioning and analyzed by electron microscopy. Oscillation X-ray diffraction data was collected on image plates using synchrotron radiation and oscillation angles of either 0.25 or 0.30 degrees. A low-resolution 6.5 A data set collected from a single frozen crystal was particularly helpful in the structure determination, because of its completeness and internal consistency. The initial particle orientations were determined using self-rotation functions, while the initial position of one particle was determined from a Patterson map. The structure was solved by molecular replacement real-space averaging using a model based on a cryo-electron microscopy reconstruction as a starting point for the phase determination. The initial structure determination used the data between 20 and 13 A resolution, which was then extended one reciprocal lattice point at a time to 6.5 A resolution. At this point, a 3.5 A resolution data set compiled from a number of crystals collected at 277 K was introduced. Phase extension and averaging continued to 3.5 A resolution after re-determining the particle positions and orientations. The amino-acid sequences of most of the D, F and G proteins and part of the B protein could be unambiguously built into the 3.5 A electron-density map. Partial crystallographic refinement yielded an R factor of 31.6%, consistent with the relatively low resolution and lack of completeness of the data.

Structure of a viral procapsid with molecular scaffolding.

The assembly of a macromolecular structure proceeds along an ordered morphogenetic pathway, and is accomplished by the switching of proteins between discrete conformations as they are added to the nascent assembly. Scaffolding proteins often play a catalytic role in the assembly process, rather like molecular chaperones. Although macromolecular assembly processes are fundamental to all biological systems, they have been characterized most thoroughly in viral systems, such as the icosahedral Escherichia coli bacteriophage phiX174. The phiX174 virion contains the proteins F, G, H and J. During assembly, two scaffoldingproteins B and D are required for the formation of a 108S, 360-A-diameter procapsid from pentameric precursors containing the F, G and H proteins. The procapsid contains 240 copies of protein D, forming an external scaffold, and 60 copies each of the internal scaffolding protein B, the capsid protein F, and the spike protein G. Maturation involves packaging of DNA and J proteins and loss of protein B, producing a 132S intermediate. Subsequent removal of the external scaffold yields the mature virion. Both the F and G proteins have the eight-stranded antiparallel beta-sandwich motif common to many plant and animal viruses. Here we describe the structure of a procapsid-like particle at 3.5-A resolution, showing how the scaffolding proteins coordinate assembly of the virus by interactions with the F and G proteins, and showing that the F protein undergoes conformational changes during capsid maturation.

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.

The capsid size-determining protein Sid forms an external scaffold on phage P4 procapsids.

Although the phages P2 and P4 build their capsids from the same precursor, the product of the P2 N gene, the two capsids differ in size: P2 builds a 60 nm, T = 7 capsid from 420 subunits, whereas P4 makes a 45 nm, T = 4 capsid from 240 subunits. This difference leads to substantial changes in shell geometry and subunit interactions. Previous results have demonstrated that the P4 sid gene is responsible for the assembly of P4-sized shells. We have used cryo-electron microscopy and image reconstruction to determine the structure of a putative assembly intermediate of P4 capsids, produced in vivo from cloned genes. We demonstrate that Sid forms a P4-specific scaffold with icosahedral symmetry on the outside of the procapsid-like particles. The Sid molecules (60 or 120 copies) form lofty arches that interact with the gpN hexamers on the icosahedral 2-fold axes, and connect as trimers over the 3-fold axes, forming a continuous dodecahedrally shaped outer cage. The gpN shell inside the Sid cage is approximately 40 nm wide, consistent with the previously suggested maturational expansion. The main difference with respect to the mature P4 capsids is found in the hexamers, which appear strongly elongated and more protruding than in the mature shell. These and previous results are discussed in the light of a model for regulation of capsid size.

Bacteriophage P2 and P4 morphogenesis: assembly precedes proteolytic processing of the capsid proteins.

Several of the structural proteins of phage P2 and its satellite P4 undergo proteolytic processing during development of mature phage particles. Here, we report that uncleaved shell protein, gpN, is present in immature capsids of both P2 and P4, showing that assembly precedes processing. This excludes the possibility that processing of gpN is involved in capsid size determination. We also find that N*, the fully processed version of gpN, produced from a plasmid, can assemble into both P2- and P4-sized particles, implying that the amino-terminal end of gpN is not required for assembly initiation nor for the formation of a T = 4 shell. As may be expected for a scaffolding protein, we find that gpO coexists with gpN in immature P2, as well as P4, capsids. This result supports the conclusion that gpO is required for both phages and strongly suggests that the O derivative, h7 (found in mature capsids), results from proteolytic cleavage after gpN/gpO coassembly.

Bacteriophage P2 and P4 assembly: alternative scaffolding proteins regulate capsid size.

The capsid protein of bacteriophage P2, encoded by the N gene, can assemble into icosahedral capsids of two possible sizes, with diameters of 60 and 45 nm, respectively. Only the larger capsid is used by P2 itself, but the smaller one is exploited by the satellite phage P4. We have analyzed the assembly products of gpN expressed in vivo from a plasmid, i.e., in the absence of any other phage proteins, and find that gpN alone forms closed shells of both sizes, although with poor efficiency. Coexpressing gpN with gpO, the putative P2 scaffolding protein, increases the efficiency of large particle formation. In contrast, introducing the sid gene by P4 infection stimulates the assembly of small particles. Our results suggest that gpO and gpSid act competitively with respect to capsid size determination. Furthermore, we demonstrate that gpN alone undergoes the normal proteolytic maturation steps, implying that gpN processing is either autocatalytic or mediated by a host enzyme.

Structural transitions during maturation of bacteriophage lambda capsids.

The three-dimensional structures of the procapsid and of the mature capsid of bacteriophage lambda were determined to a resolution of approximately 3.4 nm by cryo-electron microscopy and image processing. The mature lambda capsid contains two major proteins, gpE and gpD, arranged on a T = 7 lattice, with gpE arranged as hexamers and pentamers and gpD arranged as trimers. The hexamers and pentamers in the virion display a cartwheel-like structure, with skewed spokes (or arms) radiating out from a central hexameric hub. The thimble-shaped gpD trimers are superimposed on the trivalent interaction point of these arms. A reconstruction of a lambda D- mutant capsid to lower resolution shows no trace of these trimers, thus revealing the interactions of the underlying arms. The procapsid has elongated, irregularly shaped hexamers with gpE subunits set perpendicularly to the capsid surface.

Characterization of the capsid associating activity of bacteriophage P4's Psu protein.

The Psu (Polarity suppression) protein of satellite bacteriophage P4 was first characterized as an anti-terminator of transcription termination in Escherichia coli. Psu is also a structural component of mature P4 capsids, where it is present as a decoration protein. Psu is located externally on the capsid surface, and it appears to protect the capsid from loss of DNA through the capsid shell. The ability of Psu to specifically bind to the P4 capsid appears not to be dependent on any P4 specific components such as the capsid protein cleavage products h1 and h2, or P4 DNA. We suggest that Psu binds to the P4 capsid as a result of the special structure of the hexamers in the P4 capsid.

Capsid localization of the bacteriophage P4 Psu protein.

In addition to its polarity-suppressing activity, the Psu protein of bacteriophage P4 also serves to stabilize the capsid against heat treatment and binds externally to the phage capsid. However, the heat stability is lost upon purification of the virus, indicating a loss of Psu protein from the capsid. By using three-dimensional reconstruction from cryo-electron micrographs of P4 psu1 amber mutants lacking Psu, and of P4 virions, which have been saturated with Psu protein to regain heat stability, we have determined the position of this protein on the virus surface. Our results are consistent with the hypothesis that the function of Psu is to stabilize the hexameric capsomer assembly.

Image reconstruction from cryo-electron micrographs reveals the morphopoietic mechanism in the P2-P4 bacteriophage system.

The satellite bacteriophage P4 does not have genes coding for any major structural proteins, but assembles a capsid from the gene products of bacteriophage P2. The capsid assembled under control of P4 is smaller (45 nm) than the normal P2 capsid (60 nm). The low resolution (4.5 nm) structures of P2 and P4 capsids were determined by cryo-electron microscopy and image processing. The capsid of P2 shows T = 7 symmetry with most of the mass clustered as 12 pentamers and 60 hexamers. The P4 capsid has T = 4 symmetry with a similar distribution of mass to P2, but the hexamer geometry has changed. The major capsid protein has a two-domain structure. The major domains form the capsomers proper, while connecting domains form trivalent contacts between the capsomers. The size determination by P4 appears to function by altering hexamer geometry rather than by affecting the interdomain angle alone.

Three-dimensional structure of the surface layer of Wolinella recta.

The three-dimensional structure of the crystalline surface layer (S-layer) of Wolinella recta ATCC 33238T, a gram-negative, anaerobic periodontopathogen, was determined to 3.8 nm resolution by electron microscopy and digital image processing. The S-layer protein is closely associated with the outer bacterial membrane, and shows p6 symmetry with lattice spacing and thickness of 21 nm and 15 nm, respectively. The funnel-shaped subunits consist of 6 heavy domains located round a common base at the sixfold axis, and communicate with the adjacent subunits through a lighter domain at the threefold axis (M6C3 arrangement).