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The three-dimensional structure of a DNA translocating machine at 10 Å resolution José María Valpuesta1, José Jesús Fernández2, José María Carazo1 and José L Carrascosa1* Background: Head–tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process. Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions. Results: Recombinant bacteriophage φ29 connectors have been crystallized in two-dimensional aggregates. An average projection image and a threedimensional map have been obtained at 8 Å and 10 Å resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals. The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass. The threedimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail. At the junction of the two domains, 12 smaller appendages are visualized. A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through.

Addresses: 1Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain and 2Departamento de Arquitectura de Computadores y Electrónica, Universidad de Almería, 04120 Almería, Spain. *Corresponding author. E-mail: [email protected] Key words: bacteriophage, cryoelectron microscopy, image processing, viral connector Received: 4 November 1998 Revisions requested: 3 December 1998 Revisions received: 21 December 1998 Accepted: 11 January 1999 Published: 26 February 1999 Structure March 1999, 7:289–296 http://biomednet.com/elecref/0969212600700289 © Elsevier Science Ltd ISSN 0969-2126

Conclusions: The propeller-like structure of the φ29 connector strengthens the notion of the connector rotating during DNA packaging. The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation.

Introduction The prohead–tail connector, or portal protein, is a key substructure of double-stranded (ds) DNA bacteriophages and has roles in several steps of the phage morphogenetic cycle. These roles include formation of the precursor capsid (or prohead), DNA packaging into the prohead, and tail binding to the mature head [1–4]. Structural information for several of these connectors has been obtained at low resolution using electron microscopy and image processing (for a review see [4]). From these studies, it can be inferred that all viral connectors have a very similar structure despite their lack of sequence homology. Doubtless, the structural similarity has to do with the role of the connector in the viral life-cycle. The bacteriophage φ29 connector is the most well studied of these structures, both functionally and structurally. The connector has several domains, some of which have been partially characterized during the past few years. Firstly, there is the prohead domain; the connector interacts (but does not bind) with the prohead and the presence of this domain is essential for correct prohead formation [5,6]. The second domain — the tail domain — is responsible for binding with the collar and tail proteins [7,8]. The third and fourth domains comprise a DNA-binding domain and an RNA-binding domain. The DNA-binding

domain seems to be important for the recognition of DNA in the first steps of DNA packaging [9]. The RNA-binding domain binds to a specific RNA of viral origin (prohead RNA, pRNA) and is essential for DNA packaging [10]. Finally, there is a domain that interacts with a specific phage ATPase, protein p16, which provides the energy necessary for the DNA translocation process to occur [11,12]. As the connector seems to have such a central role in the viral life-cycle, providing the physical base for a number of key morphogenetic interactions, any structural information obtained could help us to understand how the connector functions are related to its structure. Here, we show an average projection image and the threedimensional reconstruction of the bacteriophage φ29 connector at 8 Å and 10 Å resolution, respectively. The data were obtained from frozen-hydrated specimens of twodimensional crystals. The three-dimensional map is the first to be obtained directly from the protein aggregate without the use of any staining agent, and allows the detection of finer structural features than those generated previously [13,14]. The structural data obtained have been used to suggest a relationship between certain observed structural features and their role in the DNA packaging mechanism.

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Results and discussion

Figure 1

The projection structure

Two-dimensional crystals of phage φ29 connectors were grown as described in the Materials and methods section. The connector is a highly charged macromolecule that needs the presence of high ionic strength to maintain its solubility. The removal of salt from the solution induces the connector crystallization, most likely in order to protect its charged domains from the solvent. Crystals formed in this way are in the form of large sheets ranging from 2–6 µm. Micrographs were taken at × 40,000 magnification at a low electron dose and show low contrast (Figure 1a). Five of the best micrographs were digitized and processed (as described in the Materials and methods section). The resolution of the five images after digital processing was 8 Å. Figure 1b shows the diffractogram of one of such image, which reveals systematic absences in the (2h+1, 0) and (0, 2k+1) spots, indicating the presence of screw axes in both directions of the unit cell. The dimensions of the unit cell were determined to be 165 Å × 165 Å, identical to those previously observed by cryoelectron microscopy [14] and atomic force microscopy (AFM) [15]. The five images were merged with data up to 8 Å resolution and a projection map was generated without any symmetry imposition (Figure 2a). Using the program ALLSPACE [14], the crystallographic group of this crystal was found to be P4212. The surface study of the same crystals [15] also confirmed that the unit cell contains screw axes in both directions, so that every connector is surrounded by another four connectors in an inverted position. The imposition of the P4212 crystallographic group (Figure 2b) does not induce any major change in the structural features of the projection map generated with P1 symmetry, which indicates the quality of the data at this resolution. From observations of both average projections, the connector motif can be described as a central mass with a strong 12-fold symmetry that surrounds a channel, with 12 smaller masses protruding from the central structure. This correlates well with the connector being composed of 12 subunits of protein p10. There has long been a debate over the symmetry of the connector and, therefore, of the number of subunits within the oligomer. Whereas some authors reported the φ29 connector as having 12-fold symmetry [13], others have described the connector as having 13-fold symmetry [16,17]. However, more recent studies carried out by cryoelectron microscopy at 8–10 Å on two-dimensional crystals have shown the presence of 12 lobes protruding from the central mass [14] (and this work). AFM performed on the same type of crystals (at ~10 Å resolution) showed, without the need for any image processing, 12 appendages around the central structure of the connector [15]. Finally, self-rotation studies performed with X-ray diffraction data obtained at 3.4 Å using three-dimensional crystals reveal a clear

Vitrified two-dimensional crystals of the bacteriophage φ29 connector. (a) High-magnification image of an untilted ice-embedded crystal. Unit-cell dimensions are 165 Å × 165 Å; the scale bar represents 1000 Å. (b) Computer diffraction pattern of the intensities of the image Fourier components after averaging and unbending of one of the best untilted images. The continuous ring shows the position of the first zero of the contrast transfer function corresponding to a defocus of 5150 Å. The dotted rings show the limits of resolution corresponding to 10 Å and 8 Å. The index of quality (IQ) value of a spot is given by the signal-to-noise ratio of its amplitude. An IQ = 7 refers to a spot where the height of the peak, after subtracting the background, is equal to the background level. IQ values of 1 to 4 are marked; weaker spots (IQ = 5 to 8) are indicated by smaller boxes without numbers.

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Figure 2 Projection maps of bacteriophage φ29 connector crystals represented as gray-level images. The figure shows an average projection map of five images (a) without symmetry imposition (P1) and (b) with P4212 symmetry imposed. The unit cell is indicated in (b) as a white box. An area of 2 × 2 unit cells is shown in each map.

12-fold symmetry for the φ29 connector [18]. All this data clearly indicate that the φ29 connector is composed of 12 subunits of the protein p10, and seems to definitively settle the controversy regarding the connector symmetry. The projection of the 12 connector lobes is very complex. As shown in Figures 2a and b, the 12 appendages from one connector motif are grouped into four groups of three lobes, the same number of groups as adjacent connectors (all of which are inverted with respect to the first one, according to the P4212 symmetry group). Each of the three lobes is different from the other two in the same group, but identical to one in the other three groups. This complex projection image is generated by the interaction between the lobes from two adjacent and inverted (with respect to each other) connectors (Figure 3a). Three lobes from each connector interact with three lobes from an opposed connector in such a way that the three lobes from the first connector are placed below those from the second. The interaction is asymmetric so that each of the three lobes interacts with the opposed lobe in a different manner (see Figure 2). A careful study of the interaction between the connectors was carried out as follows. A rotational power spectrum of the connector generated using P1 symmetry was performed. The spectrum generated for the whole particle (central mass and lobes) indicated that 38% of the rotational energy belonged to the 12-fold symmetry (Figure 3b; this percentage was 47% if only the central mass was taken into account). As an approximation to the real structure of the connector, an image was generated by imposing the 12-fold symmetry onto one of the connector motifs (Figure 3c). The 12-fold symmetrized connector shows a clear handedness that was previously observed in the AFM study of two-dimensional crystals and which will be discussed later. To study the interaction between

two adjacent connectors, a synthetic projection image was generated with two 12-fold symmetrized connectors (one with the hand inverted with respect to the other) that interacted at a fixed distance between the center of the two connector channels (the same distance as measured in the projection maps of Figure 2). The two connectors were then rotated by 30° (360°/12) in 1° steps (each one in opposite directions) to find the position in which the projected image of the two symmetrized connectors coincided with that of two connectors of the average projection image shown in Figure 2a (a detail is shown in Figure 3d). The position that fits best with the average image is the one in which the first lobe is located 20° upwards (in relation to an imaginary axis running between the center of the channels of the two opposed connectors), and the other two lobes are located 10° and 40° downwards (Figure 3e). This position not only mimics the projection of the average image, but is also the one that maximizes the area of interaction between the lobes of the two connectors. The three-dimensional map

From several hundred micrographs, 50 images with tilt angles ranging from 0° to 45° were selected, processed and combined in the P4212 crystallographic group to generate a three-dimensional reconstruction of the φ29 connector. Although the data go further than 10 Å resolution for the images with 0–20° tilt, this is not the case for images with higher tilt angles (the resolution of the images at 40–45° tilt was 12–15 Å in most cases) and so the data were truncated at 10 Å. The resolution obtained in the final map was calculated to be 10 Å in the xy* direction and 17 Å in the z* direction (see Materials and methods section). The merged data contained 7210 unique reflections with an index of quality (IQ) ≤ 6, and an average phase residual of 15° between 200–20 Å, 22° between 20–15 Å and 32° between 15–10 Å.

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Figure 3 The projection of the lobes from interacting connectors. (a) Cartoon illustrating the proposed interaction between adjacent connectors. (b) Rotational spectra (from harmonics 2 to 15) of a whole connector motif extracted from the P1 projection map (continuous line) and the central mass from the same motif (dotted line). (c) The projection structure of a connector motif extracted from a P1 projection map after applying 12-fold symmetry. (d) A detail of the P1 projection shown in Figure 2a. (e) The projection structure of two 12-fold symmetrized connectors (one inverted with respect to the other) interacting at the angle which maximizes the area of interaction between the lobes of the two connectors.

Several views of the three-dimensional map are shown in Figure 4. The structure maintains the general features observed in a previous reconstruction obtained from negatively stained specimens [13], although many more details begin to emerge. The connector has a height of 75 Å and comprises two domains: a wide domain (135 Å wide × 40 Å high) that is inserted in the prohead [19,20], and a narrow domain (62 Å wide × 24 Å high) that is known to interact with the collar proteins and the tail [8,21]. The wide domain is comprised of a central structure (80 Å wide) surrounded by 12 appendages, each having a width of 27 Å. The central structure ends in 12 long rods of 15 Å in height, which were previously observed by AFM studies [15]. These rods surround a channel that connects the wide and narrow domains. The channel has a cup shape with the diameter varying from 58 Å in the wide domain to 33 Å in the narrow domain. The channel has been found to be closed in this narrow region after the DNA has been packaged into the prohead [7,8,20], whereas in the case of the phage T3 connector the channel is closed between the wide and narrow domains [22,23]. In φ29, at the junction between the narrow and wide domains, 12 small appendages protrude from the central structure,

generating a ‘valley’ between each of these appendages and the wall of the narrow domain. At the bottom of the valley, 12 ‘windows’ connect the outer surface with the interior of the channel. The connector possesses a clear vorticity that might have a role in the packaging of DNA. Several different models have been suggested for the role of the connectors from dsDNA bacteriophages in DNA packaging [2,4,24,25], but all involve the rotation of the connector. This rotation is based in the symmetry mismatch between the vertex of the prohead and the connector, as first suggested by Hendrix [26]. The vertex of the prohead in which the connector is inserted has a fivefold symmetry, whereas all the connectors with known symmetry (i.e. those of the bacteriophages φ29, λ, T4, P22, T3/T7, SPP1 and P2/P4) have either 12- or 13-fold symmetry, and therefore could rotate taking advantage of the low potential barrier governing this rotation. How do the connector and prohead interact during the rotation process? Early work by Driedonks and Caldentey [19] and the recent three-dimensional reconstruction of the φ29 bacteriophage [20] show that the wide domain of

Research Article Bacteriophage φ29 connector Valpuesta et al.

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Figure 4 Surface-shaded three-dimensional representation of the bacteriophage φ29 connector. (a) Several views are presented. (b) A cartoon depicting the hypothesized location of the connector within the prohead vertex. The vertex fits into the groove formed by the large and small appendages, so that although the connector can rotate its liberation inside or outside the prohead is prevented.

the connector is inserted into the prohead so that the appendages interact with the prohead inner surface. On the other hand, the narrow domain protrudes from the prohead and can interact with the collar and tail proteins [21]. The three-dimensional propeller-like structure found for the φ29 connector (i.e. a wide domain surrounded by a set of appendages and a narrow domain with a channel running through both domains) is also observed in other connectors, such as those from T3 [23] and SPP1 [27], in spite of the lack of any apparent sequence homology. Therefore, there must be a relationship between the structural features observed in the connector and their role regarding its interaction with the prohead. The large appendages of the wide domain prevent the liberation of the connector and may act as ball bearings during the connector rotation. However, while the appendages prevent the connector from being liberated outside the prohead, there is apparently no analogous mechanism to prevent the connector liberation inside the prohead before or during the DNA packaging. The 10 Å resolution threedimensional map shown here reveals 12 small protrusions between the wide and narrow domains that could have this role. Thus, the border of the prohead vertex would fit into the groove formed by the large and small appendages

and the connector rotation would be guided without any loss of efficiency caused by a possible rocking of the connector during its rotation. These appendages do not seem to be present in the three-dimensional reconstructions of the phage T3 [23] or SPP1 connectors (although a small hint of a protrusion is observed in SPP1; see Figure 1 in [27]). The absence of these appendages may be a problem of resolution, however, as the appendages observed in the 10 Å resolution three-dimensional reconstruction of the φ29 connector were not visible in the previous reconstruction carried out at 18 Å with negatively stained specimens [13] but only began to emerge in reconstructions carried out at intermediate resolutions (JMV, JJF, JMC and JLC, unpublished results). In spite of the efficient mechanism of connector rotation described, the packaging of DNA into the bacteriophage prohead is an energy-consuming process that needs the presence of an ATPase [2,4,24] to translocate a highly charged molecule of DNA against a concentration gradient. In the case of bacteriophage φ29, the DNA molecule is ~ 850 times longer than the connector itself (and much larger in other bacteriophages). The propeller-like shape of the φ29 connector strengthens the notion of its rotation and

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the vorticity of the structure suggests that the rotation is always performed in the same direction. In this way, the connector translocates the DNA through a mechanism that is intimately related to its structure. How would this unidirectional rotation of the connector be accomplished? The answer must lie in the concerted action of the ATPase and connector. There is clear evidence for several of the bacteriophages studied, that the connector and ATPase interact during the packaging process (see [4] and references therein). The chemical energy generated during ATP hydrolysis must, therefore, be directly transmitted to the connector. Several ATPase molecules may bind to the connector oligomer (the page φ29 ATPase, p16 protein, binds to the connector in a ratio of approximately 12:1 [28]). An important point, at least in the case of bacteriophages related to phage φ29, is the presence of the prohead RNA (pRNA), that is essential for the packaging of DNA. It has been shown recently that six molecules of pRNA form a hexameric ring that may encircle the connector structure and serve as part of the DNA translocation machinery [29,30]. It has been suggested that alternating contraction and relaxation (driven by ATP hydrolysis) of the pRNA protrusions of the hexameric ring might be used to induce the rotation of the connector and therefore the DNA translocation into the prohead [30]. This hexameric ring may be located around the tip of the narrow domain of the φ29 connector [31] (M Valle, L Kremer, C Martinez–A, F Roncal, JMV, JP Albar and JLC, unpublished results). The three-dimensional reconstruction of the bacteriophage φ29 connector reported here, although at insufficient resolution to detect any hint of secondary structure, offers useful information to suggest a relationship between certain structural features observed in the connector and their role in the DNA packaging mechanism. As a result, it is possible to hypothesize several aspects of the connector working machinery.

Biological implications The structural information obtained from a variety of bacteriophage prohead–tail connectors points to a similarity in their structure that is related to their various common functions, such as prohead formation, DNA packaging, tail binding to the mature head and bacteriophage infection. The 10 Å resolution three-dimensional map of the bacteriophage φ29 connector, presented here, shows some structural features that help us to understand how the connector interacts with the prohead and sheds some light on its role during DNA packaging. The φ29 connector, a dodecamer of protein p10, contains two large domains: a wide domain that is inserted into the prohead and a narrow domain that protrudes from the prohead and interacts with the tail. A channel runs along these two domains that is wide enough to allow a

double-stranded DNA molecule to pass through. Twelve lobes protrude from the wide domain, generating a propeller-like structure similar to that previously observed in other connectors. The mismatch of symmetry between the connector structure (12-fold) and the prohead vertex (fivefold) suggests that the connector acts as a DNA rotary motor (the energy being provided by an ATPase) that relates its movement to the translocation of DNA. The 12 lobes from the wide domain and 12 smaller lobes located between the wide and narrow domains form a groove in which the prohead vertex may fit so that the connector could rotate without being liberated either inside or outside of the prohead. The 12 large lobes may act as ball bearings interacting with the inner wall of the prohead during the connector rotation.

Materials and methods Protein purification and crystallization

The connectors of φ29 were overexpressed in Escherichia coli carrying a recombinant plasmid containing the gene encoding for protein p10 (molecular mass 35,800), of which the φ29 connector is composed [32], and purified as described previously [33]. Crystals of φ29 connectors were obtained by incubating a solution of the purified connectors (3–4 mg/ml) in 2 M NaCl at 4°C and abruptly decreasing the ionic strength by dialysis against distilled water. Thin crystals with an average size of 2–4 µm were obtained within a few hours.

Electron microscopy and specimen preparation Grids were prepared by evaporating carbon on to freshly cleaved mica, which was then floated on water and lowered on to the grids. These were glow-discharged in air or N-amylamine before being used. Samples of φ29 connector crystals (1–2 µl) were applied to the carbon film for 3 min, blotted for 15–20 s and frozen quickly in liquid ethane at –180°C, so that the crystals were preserved in vitreous ice. The frozen grids were kept under liquid nitrogen prior to use. Electron micrographs were obtained using a JEOL 1200EX-II operated at 120 kV and recorded on Kodak SO-163 film. The images were normally obtained with a nominal magnification of × 40,000 and underfocus from 4000 to 9000 Å. A GATAN-626 cold stage was used in the recording of the electron micrographs from specimens at temperatures below –160°C.

Image processing Micrographs were screened by optical diffraction to select those suitable for further processing on the basis of the presence of reflections at high spatial frequency. Square areas of 6000 × 6000 or 5000 × 5000 pixels of the selected micrographs were digitized on a Perkin-Elmer 1010 M flat-bed microdensitometer at 10 µm step size, corresponding to a 2.5 Å sampling rate at the specimen level. The digitized areas were subjected to a structural analysis according to the approach previously described [34] in an attempt to discover structural heterogeneities within the crystalline areas that would preclude the electron crystallography methodology. This approach combines the use of local-averaging, artificial neural networks and multivariate statistical analysis and was carried out using the XMIPP program suite [35]. This analysis was performed to choose the micrographs used for the projection map and for the three-dimensional reconstruction. A combination of software packages was used to take advantage of the unique capabilities of each. Most of the programs belonged to the MRC image processing suite [36]. Lattice refinement was carried out using the X-windows-based graphical environment SPECTRA [37]. Next, lattice distortion correction was performed by the methods described in [38]. Two rounds of lattice unbending were sufficient for

Research Article Bacteriophage φ29 connector Valpuesta et al.

most of the images processed, although some improvements were obtained in a few cases by applying some additional unbending steps. The contrast transfer function (CTF) affecting each image was detected by using an approach previously described [39]. In essence, the image was divided into many small areas, their Fourier transforms calculated and, finally, the amplitudes of these Fourier transforms averaged. With this approach, the detection of the CTF oscillations (Thon rings) is greatly facilitated. In the case of tilted images, the small areas in which Fourier transform amplitudes were to be averaged were only those located along the tilt axis. In this way, the defocus at 0° tilt was estimated. The graphical tool employed to determine the CTF parameters, in either the untilted or tilted case, was the integrated crystallographic environment (ICE) [40]. The correction for the CTF effects was performed using the programs CTFAPPLY and TTBOX (the latter being used for images with tilt angles of higher than 10°) [41].

Projection structure analysis Five untilted images were selected and merged using the program ORIGTILT from the MRC suite [36] and the previously determined crystallographic space group P4212 [14]. Only reflections with IQ ≤ 6 were used in the phase origin refinement. The resulting amplitudes and phases were averaged using the program AVRGAMPHS (MRC suite). Finally, a Fourier synthesis was obtained from this set of averaged amplitudes and phases. The resolution of the projection was determined by the procedure described in [38]. The projection map was subjected to a rotational power analysis [42]. When needed, a 12-fold rotational filtering consisting of superimposing rotated versions of the structure at 360°/12, was applied to the connector motif [43].

Three-dimensional reconstruction As described previously, 50 images with tilt angles ranging from 0° to 45° were selected. The nominally untilted image data were first combined and used as a reference set. Images were subsequently merged in order of increasing tilt angle following the ideas developed in [44]. Merging was performed as described above using the space group P4212 [14]. Prior to the merging, the tilt angles and axes of each image were calculated according to the procedure described in [45]. A refinement of the tilt angle and tilt axis was later necessary for the images with tilt angles lower than 20°. The program LATLINE [46] was used to fit smooth amplitudes and phase curves to the non-uniformly sampled data along each reciprocal lattice line. Sampling the curves at uniform intervals provided the amplitudes and phases used for the subsequent three-dimensional structure computation. The choice for the uniform sampling interval along the lattice lines was a third of the inverse of the unit-cell thickness, according to [44]. The unit-cell thickness was properly calculated by AFM in previous work [15] as ~100 Å. The CCP4 suite of programs [47] were used to calculate the three-dimensional structure of the φ29 connector from the uniform samples taken along the lattice lines at intervals of 1/300 Å–1. Figure of merit weighting was applied to downweight unreliable structure factors, as described [48]. The final sections of the three-dimensional map were 12-fold symmetrized to separate the appendages from overlapping connectors. A volume was then generated using these sections. The resolution of the final structure was determined according to the procedure described in [49], which consisted of estimating the threedimensional point spread function (PSF) derived from the quality measurements of the samples in the three-dimensional Fourier distribution. The effective resolution in each axis (x,y,z) is then given by the width of the PSF at half height in the axis considered. An additional scale factor is needed to translate that effective resolution in terms of cut-off frequency. The visualization of the surface-rendered reconstructed volume of the φ29 connector was carried out using AVS (application visualization system) [50].

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Acknowledgements The help of Pilar Vicente with the scanning of the micrographs is acknowledged. This work was partially supported by grants PB96-0818 (JLC), PB97-1225-C02 (JMV), BIO95/0768 and BIO98/0761 (JMC) from the Dirección General de Investigación Científica y Técnica.

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