Structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor, P

Abstract

The negative-strand RNA viruses (NSRVs) are unique because their nucleocapsid, not the naked RNA, is the active template for transcription and replication. The viral polymerase of nonsegmented NSRVs contains a large polymerase catalytic subunit (L) and a nonenzymatic cofactor, the phosphoprotein (P). Insight into how P delivers the polymerase complex to the nucleocapsid has long been pursued by reverse genetics and biochemical approaches. Here, we present the X-ray crystal structure of the C-terminal domain of P of vesicular stomatitis virus, a prototypic nonsegmented NSRV, bound to nucleocapsid-like particles. P binds primarily to the C-terminal lobe of 2 adjacent N proteins within the nucleocapsid. This binding mode is exclusive to the nucleocapsid, not the nucleocapsid (N) protein in other existing forms. Localization of phosphorylation sites within P and their proximity to the RNA cavity give insight into how the L protein might be oriented to access the RNA template.

Vesicular stomatitis virus (VSV) belongs to the family Rhabdoviridae, which also includes rabies virus. The rhabdoviruses are part of the broad group of negative-strand RNA viruses (NSRVs), which contain many medically relevant viruses, including avian influenza, measles, and Ebola. VSV has long served as a prototypic nonsegmented negative-strand RNA virus (NNSRV), partly because of the small number of genes that are encoded by its 11-kb genome (1). These genes include the nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and a large polymerase protein (L). Each of these proteins has multiple functions and, as a result, has multiple binding partners, including but not limited to each other.

The NNSRVs are characterized by the unique fact that in the entire replication cycle, their genomes do not exist as naked RNA, but rather are encapsidated by their nucleocapsid proteins. The nucleocapsid is the active template for transcription and replication (2, 3). Structures of the nucleocapsid-like particles (NLPs) of VSV and rabies virus have recently been solved (4, 5), showing that the N protein has 2 lobes angled together to form a cavity for encapsidation of the genomic RNA. Each N monomer accommodates 9 bases of RNA. The structures also revealed that each monomer of N interacts with 3 neighboring N molecules across the nucleocapsid. The contacts involve the elongated N terminus and an extended loop (C loop) within the C-terminal lobe, and they are required for RNA encapsidation (6). Residues within this loop have also been implicated in binding to the P (7, 8). Recently, more insight into capsid formation was gained through crystallographic studies of an N protein with a serine-to-tryptophan mutation at residue 290 (called N290; ref. 6). N290 has lost the ability to encapsidate RNA because of the bulky side chain of tryptophan in the RNA cavity, yet the capsid assembly functions of the protein remain intact. Thus, the N protein alone contains all of the information for the assembly of a capsid structure.

The multidomain P protein (Indiana strain) of VSV is the 265-aa nonenzymatic cofactor of the viral polymerase. The N-terminal domain comprises the first 106 residues of P and contains 3 residues (Ser-60, Thr-62, and Ser-64) that may be phosphorylated by host casein kinase II (9–11). These residues are indispensable because they are required for transcription (12–14). The central domain, residues 107–177, is the site of P dimerization and subsequent tetramer formation (15, 16). Previous experiments have shown that multimerization beyond the dimeric state is essential to P's role in replication (11, 17). The majority of the remaining 89 residues form the C-terminal domain (P_CTD). P_CTD contains 2 additional phosphorylation sites (Ser-226 and Ser-227). The L protein has been shown previously to interact with these residues (13), and although phosphorylation is not required for L binding, phosphorylation of either residue is responsible for regulating levels of replication. This domain of P is also the major site of N protein association (18–23). The structure of the P_CTD was determined recently by NMR and shown to form a single compact unit comprising an antiparallel β-turn and 5 α-helices (24).

Upon translation and before polymerization of N onto the genome, N forms an initial complex with P, known as N^o-P (25–27). This RNA-free, encapsidation-competent complex is delivered to the active replication site, possibly via the secondary interaction of P with the viral polymerase. Here, N comes into contact with the newly synthesized genomic RNA, and the process of encapsidation occurs. In addition to binding N and forming the encapsidation precursor, P binds to the nucleocapsid during the viral replication cycle that involves polynucleotide synthesis. The L polymerase subunit cannot recognize the nucleocapsid alone. The processes of transcription and replication require the association of the P as a component of the viral polymerase complex (2, 28). Thus, P is the determining factor for template recognition for the viral polymerase.

The N, P, and L proteins are unique in that they form distinct complexes at different stages of the replication cycle. As described above, L and P form the RNA polymerase that associates with the N-enwrapped template. Additional experimentation has shown that some P mutants maintain the ability to bind N and form N^o-P but are inactive in transcription. These complexes were, however, capable of supporting replication (22). Therefore, it was surmised that the replicase and transcriptase were 2 distinct complexes. Subsequently, an active replicase complex was isolated, and in fact it was shown to contain the N, P, and L proteins (29). This tripartite complex was distinct from the transcriptase, a complex that does not contain the N protein. These experiments confer the complexity of the interactions of N, P, and L, and although structures of N and 2 individual domains of P have been published, the precise nature of the direct interaction of N and P together is unknown. Likewise, the nature of the tripartite complex between N, P, and L is not well understood. Ultimately, because these complexes play key roles in the synthetic processes of transcription, replication, and encapsidation of progeny genomes, atomic-level snapshots of their interactions would be invaluable in aiding interpretation of previously published works.

To date, a large body of work has been performed to aide in understanding how the P interacts with N. These studies have relied heavily on mutagenesis and biochemical analysis. However, a concrete model of the direct interaction of N and P was still elusive. The work presented here gives the atomic look at the complex formed between the N protein in NLP and the C-terminal nucleocapsid-binding domain of P. Two adjacent monomers within the nucleocapsid form a unique binding site for P, which is a binding site that can only be found in the nucleocapsid. Binding occurs solely to the C lobes of the 2 N proteins. This explains how the P delivers the viral polymerase specifically to the nucleocapsid for RNA synthesis. New insights are found in the mechanism by which the P allows the polymerase complex to recognize the nucleocapsid as the template for viral RNA synthesis.

Results

N/P_CTD Structure.

The N protein is present in the nucleocapsid as a linear polymer. The P protein must recognize the polymeric N protein template and, subsequently, P must remain associated with the nucleocapsid as the polymerase complex moves down the template during RNA synthetic events. These 2 events are essential to the function of the viral polymerases of the NNSRV. To date, it has been unclear exactly how the N protein and P associate. The structure of the middle multimerization domain of P (residues 107–177) was reported previously as a dimer (15). It was also reported recently that the structure of the uncomplexed C-terminal 71 aa of P (residues 195–265) contains a single compact domain comprising an antiparallel β-turn and 5 α-helices (24). Here, we present the crystal structure of P_CTD in complex with NLP in the presence and absence of RNA. There are no significant differences between the 2 structures. The overall rmsd between the RNA-encapsidated P_CTD complex and the RNA-deficient N290 P_CTD complex is 1.41 Å. When superimposed, the main noticeable difference is the orientation of the N lobe with respect to the center of the 10-member ring. These differences were also observed in NLP structures in the absence of P_CTD (4, 6). In the NLP–P_CTD complex, N exists as a decameric ring with all intramolecular interactions intact, as in the uncomplexed NLP (4, 6), whereas each monomeric P_CTD interacts with 2 adjacent N monomers (Fig. 1). This is an important feature because this unique binding site for P could be present only in the nucleocapsid. A total of 18 residues from P_CTD contribute to the interaction with the dimeric N-binding site. N donates 17 aa to the interaction: 11 and 6 residues from each N of the binding site, respectively. The residues from N are from a contiguous stretch, between 354 and 386, that encompasses helix α13 and the C loop, which rises above the upper surface of the C-terminal lobe. Twenty-four hydrogen bonds are formed between P and the 2 N monomers. The complete list of bonding partners is provided in Table 1. Two residues of P, Arg-260 and Lys-262, which have been implicated previously in N protein association and mutation of these residues, have also been shown to affect transcription (13). These basic residues are bonded to 2 acidic residues, Asp-358 and Glu-377, which are found on different molecules of the N protein-binding site. Interestingly, Tyr-256 and Asn-257 of P are unique, because each interacts with both molecules of N. The total surface area buried by the interactions between the P_CTD and the 2 adjacent N molecules is 956 Ų, whereas the total surface area of the P_CTD is 5,100 Ų. Thus, ≈19% of the available surface area of P_CTD is in complex with the N protein(s).

Fig. 1.

Ribbon representations of the VSV nucleocapsid in complex with the C-terminal N protein-binding domain of the P protein (P_CTD). (A) Three assembled N molecules are represented in red, green, and blue. P_CTD, shown in yellow, binds to the extended loop (C loop) within the C lobe of 2 adjacent N molecules (red and green). (B) Scaled-up image of the interaction. Residues of the dual N-binding site that are in contact with P_CTD are shown. The illustrations found in this and the subsequent figures were generated with PyMOL (41).

Table 1.

The binding interactions between the residues of the P_CTD and the residues of the dimeric N protein binding site

Interaction	P residues	Atoms	N residues (subunits 1 and 2)	Atoms
Hydrophobic	LEU214	Side chain	THR361(1)	Side chain
H bond	GLN215	NE2	ASP359(1)	OD1
H bond	GLN215	OE1	SER360(1)	N
Hydrophobic	LEU217	Side chain	LEU364(1)	Side chain
Hydrophobic	ILE219	Side chain	LEU364(1)	Side chain
H bond	SER233	O	THR365(2)	OG1
H bond	VAL234	O	LEU364(2)	N
Hydrophobic	GLY235	CA	LEU364(2)	Side chain
H bond	ARG251	NH1, NH2	THR365(2)	OG
H bond	LYS253	NZ	ASN367(1)	ND2
H bond	Lys254	NZ	ASP384(1)	OD2
H bond	Lys254	NZ	ASP384(1)	O
H bond	Lys254	O	ASN386(1)	ND2
Hydrophobic	LEU255	Side chain	THR366(1)	Side chain
H bond	TYR256	OH	ASP358(2)	O
H bond	TYR256	N	ASP384(1)	OD1
H bond	ASN257	ND2	ASP359(2)	OD1
H bond	ASN257	ND2	ASP359(2)	OD2
H bond	ASN257	ND2	LYS354(2)	NZ
H bond	ASN257	ND2	GLU383(1)	OE2
H bond	ASN257	OD1	ASP359(2)	OD1
H bond	ASN257	OD1	ASP359(2)	OD2
H bond	ASN257	OD1	SER360(2)	OG
H bond	ASN257	OD1	ASP384(1)	OD1
H bond	ASN257	N	ASP384(1)	OD1
H bond	GLN258	N	ASP384(1)	OD1
H bond	ARG260	NH1	ASP358(2)	OD1
Hydrophobic	VAL261	Side chain	VAL367(1)	Side chain
H bond	LYS262	NZ	GLU377(1)	OE2
H bond	TYR263	OH	GLY362(1)	N

The numbers 1 and 2 shown in parentheses adjacent to N protein residue names and numbers refer to molecule 1 or 2, respectively, of the N dimer. The list of residues was compiled with the aid of the Protein Interfaces, Surfaces, and Assemblies (PISA) server (42) and visual inspection with COOT (36).

The conserved hydrophobic core of P_CTD was described previously (24). Two adjacent surfaces of the P_CTD present interesting conserved hydrophobic cavities. The first hydrophobic cavity is positioned between the β-turn and 2 helices (α3 and α4), and the second is situated between helix α1 and the loop connecting helices α2 and α3. The relevance of each cavity is unknown and has been postulated as a potential binding site for the other VSV proteins. The complex structure between N and P_CTD showed that the first cavity is capped by the C loop of the N protein. The loop does not penetrate the cavity, but it does lie over the entrance. The second cavity is distal to the N-binding surface of P_CTD and is left exposed. This suggests that these hydrophobic cavities most likely form a hydrophobic core that renders stability to this small domain. However, the second cavity is solvent-accessible and could be available to interact with the domains of P that are missing from this structure, or an alternate viral or host protein.

P_CTD Structure Changes.

The topology of the P_CTD structure was described in a recent report by Ribeiro et al. (24). Although this domain forms a compact unit, the individual secondary structure elements appear to have some flexibility. It is very likely that P_CTD undergoes some conformational changes, which may not be global, upon binding to the nucleocapsid. P_CTD in the NLP–P_CTD complex is topologically identical to that described in the previous study. The size of the recombinant P_CTD used in this study was slightly longer. To show structural changes in P_CTD upon N binding, the complexed and uncomplexed P_CTD structures were superimposed. A single composite structure was created from the 20 lowest-energy deposited NMR structures with CNS (30). Superposition of the composite structure with the N-bound structure resulted in an overall rmsd of 2.16 Å for all atoms. By contrast, the rmsd between the best representative conformer in the ensemble P_CTD structure (as defined in ref. 24) and P_CTD structure from this study was 2.06 Å. In either case, each of the secondary structure elements and the overall topology of the protein are maintained upon binding the N protein. There are, however, localized shifts of the secondary structure elements, as illustrated in Fig. 2. These displacements correspond to N-binding regions of P, with the most notable shifts occurring at helix α2 and the loop connecting with helix α3 and the β-turn (Fig. 2).

Fig. 2.

Structural overlay of the VSV P_CTD in the N protein-bound state (yellow) and the unbound state (cyan; PDB accession ID: 2k47). Residues of P_CTD that make contact with the N protein have been shaded magenta on the N-bound representation. (A) Shown is the structure of the P_CTD, with secondary structure elements labeled. (B) The overlay of the 2 structures is shown.

Changes in the N Protein upon P Binding.

Upon P binding, the NLP maintains the intermolecular N interactions described previously (4, 6), with very little discernible difference in their overall structures. The main dissimilarity is in the C loop, as shown in Fig. 3. Because there seem to be structural changes of N side chains in the NLP–P_CTD complex, we used the higher-resolution structure of N290 NLP in complex with P_CTD to identify any changes in N. N290 is an N protein with a mutation of serine to tryptophan at residue 290 (6). This mutated N has lost the ability to bind RNA. Superposition of the N290 structure with the P_CTD-bound structure determined here revealed that the C loop is shifted, with many of the side chains rearranged to accommodate binding of the P_CTD (Fig. 3). The remainder of the N290 structure superimposed quite well with the P_CTD-bound structure. The 5 monomers overlaid as a single rigid body had an rmsd of 1.117 Å. If the loops (residues 256–369) were removed from the calculation, the rmsd was lowered to a value of 1.075 Å. By comparison, the rmsd for the N RNA structure to its P_CTD-bound counterpart was 1.223 Å, or 1.066 Å with the loops removed. Interestingly, in the absence of P_CTD, the extended loops were disordered in 3 of the 5 N monomers in the previously determined N RNA structure. In the P_CTD-bound structure presented here, all 5 of the loops were observed in the electron density maps and were easily traced. In this case, P_CTD promoted ordering of the loop. The inherent flexibility of the loop could aid in snaring P because P delivers the L protein to the nucleocapsid for RNA synthesis.

Fig. 3.

Structural similarity of the N protein in the P_CTD-bound and unbound states. Two molecules of the P_CTD-bound N290 molecules are colored red and green. Two additional molecules (colored gray) of the previously determined structure N290 (PDB accession ID: 2qvj) were superimposed upon the current complex structure. The main dissimilarity between the structures is observed in the conformation of the C loops (acknowledged with arrows). This corresponds to the site of P_CTD binding. The models are rotated 180° with respect to Fig. 1; in this case, looking from the exterior of the protein rather than the RNA cavity face of N.

Discussion

Polynucleotide synthesis is an essential part of the viral replication cycle. The NSRVs have evolved to perform this reaction with their own specialized polymerase proteins. For the nonsegmented NSRVs, including VSV, this requires a concerted set of events involving the N protein-enwrapped genome, the L protein, and the nonenzymatic P. One of the roles of P is to deliver L to the active template. The L protein is unable to bind the template directly but, rather, binds to the P and is then delivered to the active template as P binds to N. The structure presented here of an NLP bound to the P_CTD shows that the N protein forms a unique binding site for P that involves 2 adjacent monomers within the nucleocapsid. This binding site is formed by a contiguous stretch of residues (354–386) that includes helix α13 and the extended loop of the C lobe, the C loop. The loop from each N protein snares P by clamping onto it from opposite sides. Such a binding site is exclusive to the assembled nucleocapsid and is in accordance with the fact that the active template for transcription and replication is the N-enwrapped genome. Comparison with the unbound NLP structure shows that N is largely unchanged upon binding P, with the most significant variation occurring in the C loops that bind to P_CTD (Fig. 3). Likewise, topologically, P is unchanged upon binding to the nucleocapsid. There is, however, a shift of the secondary structure elements in relation to one another—the 2 most notable movements are helix α2 and the loop connecting to helix α3 and the β-turn (Fig. 2). Each of these elements is involved in N-binding. It is possible that the induced conformational changes in N and P allow the two to be associated more tightly when P binds to the nucleocapsid.

The structure of the NLP shows that the RNA is encapsidated in a cavity located between the 2 lobes of the N protein. During transcription and replication, the L protein must be positioned at the mouth of this cavity to gain access to the genome. The L protein has been shown previously to interact with 2 essential phosphorylation sites (Ser-226 and Ser-227) on P (13). These residues reside in the C-terminal domain of P and are observed in our structure, but they are not phosphorylated. L binding to P is not dependent on their phosphorylation, but phosphorylation of either residue is responsible for regulating levels of replication. Ser-226 and Ser-227 are found on the loop that connects 3₁₀-helix 1 and helix α2 (Fig. 4). The loop is positioned such that these residues face the interior of the N protein ring. Neither serine makes contact with the N protein but, rather, each sits exposed ≈50 Å directly above the entrance of the RNA encapsidation cavity and aligned with the interior face of the C lobe of the N protein. The interaction of L with the 2 phosphorylated serine residues would not prevent P_CTD in the P–L complex from docking on the nucleocapsid. At the same time, P_CTD could bring L in close contact with the RNA, because the 2 serine residues may be viewed as the boundary of contact between L and N. Because of the way that the RNA is sequestered while encapsidated, the N protein must undergo some conformational adjustments in order for L to gain access to the genome. How this occurs is unknown at this point. One likely scenario may involve the N-terminal 22 residues of the N protein. These residues form an arm that extends to the C lobe of a neighboring N within the nucleocapsid and holds the 2 lobes of N in the proper orientation for the formation of the RNA-encapsidation cavity. Previous mutational studies showed that if the N-terminal arm is removed, the ability to bind P remains intact, but the ability to retain RNA is lost (6). Interestingly, the integrity of the assembled nucleocapsid is not completely broken. This feature is important because it dictates that the N-terminal arm can dissociate to expose the RNA locally without affecting the global association of the nucleocapsid. The L protein or the L–P complex could cause this local dissociation, exposing the RNA in the course of polynucleotide synthesis. P binds to the extended loop of the C lobe of N, and the opposite face of the loop is in contact with the N-terminal arm of N. Upon the P–L complex association with the nucleocapsid, it could be possible that the loop–arm interaction is destabilized, causing N to open. Subsequently, after transcription or replication has occurred, the arm is repositioned, and the RNA is again stored in the cavity.

Fig. 4.

Phosphorylation of the P protein. Cartoon models of 3 molecules of the N protein (colored as in Fig. 1) are shown with encapsidated RNA (shown in stick representation). A semitransparent surface covering the 3 molecules of the N protein is shown in a shade of light blue, with the RNA cavity colored a darker shade of orange. The bound P_CTD is shown in yellow. Residues that are phosphorylated are shown as space-filling spheres and are labeled accordingly. The perspective is the same as that in Fig. 1.

Here, we propose a model for the process of polynucleotide processivity based on the structure described in this work. For L to encounter the viral genome–nucleocapsid template, the dimeric P is required. This dimer is associated with both L and N. Within the dimer, 1 P_CTD associates with the nucleocapsid. To process through the genome, N is forced to open temporarily and expose the RNA as the polymerase passes along. The mechanism for forcing the conformational change resulting in the opening of N is due to a local destabilization event caused by P–L binding. Once the binding occurs, N is opened, exposing the RNA; however, upon exit of the active bubble of polynucleotide synthesis, N returns to the closed state (illustrated in Fig. 5). The P_CTD, as shown in the previous figures, binds to the C lobe of 2 adjacent N monomers within the nucleocapsid. The association should be fleeting as the polymerase complex moves along the template. Thus, P_CTD is expected to repetitively bind and release the template as the polymerase complex moves down the template. In this scenario, the dimer is always associated with L and intermittently associated with N.

Fig. 5.

Model of the interactions of P and L with the encapsidated template. The N, P, and L proteins are labeled. Polarity of the template and progeny RNA (both shown as a yellow ribbon) are also labeled with 5′ and 3′. The P dimer in the viral polymerase delivers the L subunit to the nucleocapsid template. The nucleocapsid-bound viral polymerase forms an RNA synthesis chamber in which the genomic RNA is temporarily unencapsidated to allow the catalytic region of the polymerase to use it as the template for RNA synthesis. As the viral polymerase moves along the nucleocapsid, the previous unveiled region of the RNA genome is reencapsidated, and the next segment becomes available for RNA synthesis. The P dimer maintains the association of the viral polymerase with the nucleocapsid throughout the process of RNA synthesis. Three successive steps are labeled A, B, and C.

There must be differences between the processes of transcription and replication. For some time, the general thought has been that the difference is the presence of a sufficient quantity of N^o-P (25–27). As amounts of this encapsidation precursor complex increase, the switch to replication is initiated. However, a different hypothesis was proposed, stating that the presence of N^o-P promotes the formation of a tripartite replicase that switches to replication (29). Multimerization of P seems to be necessary, because 2 previous studies have shown that both the deletion of residues 191–210 of P or addition of an artificial peptide with this sequence affects multimerization and, subsequently, transcription and replication (16, 31). This region of P is observed in our structure and is not in contact with the N protein. In our model a single C-terminal domain of P is required for recognition of the nucleocapsid. Multimerization of P may be through the central domain region, as shown by our previous structure of the central domain of P, which can form a dimer or tetramer (15). Further structural studies of the complete polymerase complex are required to fully understand the direct interaction of L with the P-bound template.

Methods

Wild-type VSV N/RNA, N (Ser-290→Trp) mutant (N290), and the P_CTD residues 183–265 were expressed in Escherichia coli and purified as described previously (refs. 6, 15, and 18, respectively). Purified protein samples of N/RNA, N290, and P_CTD [or selenomethionine-substituted P_CTD (Se-P_CTD)] were concentrated to 11, 9, and 12 mg/mL. N (or N290) was mixed with P_CTD (or Se-P_CTD) at a 1:1.2 molar ratio. N290/Se-P_CTD crystals were grown by the hanging drop vapor diffusion method in 24-well VDX plates (Hampton Research) at 22 °C (32).

Crystals of the N/RNA–P_CTD complex were grown by the hanging drop method at 4 °C. Crystallization drops were formed by mixing equal volumes of protein with reservoir solution containing 7% PEG 4000, 250 mM NaCl, and 100 mM citrate buffer, pH 5.6. This crystal form was cryoprotected with reservoir solution containing a final concentration of 20% PEG-4000 (Hampton Research) and 20% glycerol. This crystal form belonged to the orthorhombic space group P21212. Maximal crystal growth occurred within 2 weeks. Data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 11-1 at a wavelength of 1.0 Å, with an oscillation angle of 0.3° and crystal-to-detector distance of 450 mm on a Mar 325 CCD (Marresearch) detector at cryotemperature.

The N290–Se-P_CTD protein complex was mixed with a 1:1 volume equivalent of reservoir solution consisting of 0.8–1.0 M K/Na tartrate, 200 mM NaCl, and 100 mM imidazole buffer, pH 8.0. Orthorhombic crystals grew within 1 to 2 weeks. Before data collection, crystals were cryoprotected stepwise to a final solution containing reservoir solution plus 20% glycerol and were flash frozen in liquid nitrogen. Data were collected at the South East Regional Collaborative Access Team (SER-CAT) BL22-ID at Advanced Photon Source (APS) at a wavelength of 0.94 Å, with an oscillation angle of 0.3° and crystal-to-detector distance of 450 mm on a MAR 325 CCD (Marresearch) detector at cryotemperature.

In all cases, raw intensity images were processed with the HKL2000 package (33), and structure factors were calculated with TRUNCATE (34) through the CCP4 program suite. Location of the N protein/RNA decamers in the N/RNA–P_CTD complex was achieved by molecular replacement with COMO (35) using the previously determined VSV N/RNA structure [Protein Data Bank (PDB) ID code: 2gic] as the search model. An initial model of the P_CTD was built with COOT (36) in 2F_o − F_c maps of the orthorhombic data. The N290/Se-P_CTD structure was solved by molecular replacement with the previously determined N290 model (PDB ID code: 2qvj). Crude placement of the P_CTD domains was performed with the aid of an intermediate VSV N/RNA–P_CTD model using the superpositioning protocols in O (37). The asymmetric unit of each crystal of the N/RNA–P_CTD or the N290–P_CTD complexes contains one-half of the decameric nucleocapsid-like particle bound to 5 P_CTD monomers. The stoichiometry of N:P is such that each available N-binding site (10:10) is occupied when averaged over the entire crystal. However, there is a reduced occupancy of the P_CTD in certain positions. The highest substitution occurs in alternating rather than adjacent N-binding positions. The lack of complete P substitution is not surprising, because N and P do not exist in a 1:1 ratio in mature virions (38). Rigid body refinement of the individual domains and extensions was carried out with REFMAC5 (39). Manual model building and real-space refinement were performed with COOT. Selenium sites were used to aid in confirmation of the constructed sequences. Restrained and TLS refinement were performed with REFMAC5. Refinement statistics are shown in Table 2. No outliers were found in the Ramachandran plot. All superimpositions for determining the differences between bound and unbound complexes as discussed in the text were carried out by using the secondary structure mapping procedure in COOT (40).

Table 2.

Data collection and refinement statistics

Complex	N290-Pctd	N/RNA-Pctd
Space group	P21212	P21212
Unit cell, Å
a	170.6	166.5
b	234.5	235.2
c	95.0	96.1
Resolution	30.0–2.70	30.0–3.50
High-resolution bin	2.80–2.70	3.63–3.50
Reflections (unique/total)	77,814/228,906	48,185/316,345
Completeness, %	73.1 (44.9)	98.3 (88.4)
I/σI	20.79 (2.10)	21.48 (4.31)
Rmerge	0.073 (0.419)	0.116 (0.434)
Rcryst	0.263	0.258
Rfree	0.296	0.323
Mean B value, Å2	74.58	28.09
Model
No. of atoms	19,495	19,455
No. of residues	2,460	2,460
No. of nucleic acid bases	0	45
rmsd
Bonds, Å	0.006	0.008
Angles, °	0.957	1.179
Ramachandran
Favored, %	98.6	96.0
Allowed, %	100.0	100.0

Values in parentheses represent values within high-resolution shells.