Protein J (2013) 32:635–640 DOI 10.1007/s10930-013-9526-x

Using cryoEM Reconstruction and Phase Extension to Determine Crystal Structure of Bacteriophage /6 Major Capsid Protein Daniel Nemecek • Pavel Plevka • Evzen Boura

Published online: 29 November 2013 Ó Springer Science+Business Media New York 2013

Abstract Bacteriophage /6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of /6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C2221. Matthews’s coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The selfrotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM recon˚ resolution and used as struction of the /6 procapsid at 7 A a model for molecular replacement. The phases for ˚ resolution were obtained by reflections at higher than 7 A phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 ˚ -resolution electron density map was of sufficient quality A to allow model building. Keywords Molecular replacement  Cryo-electron microscopy  Non-crystallographic symmetry  Virus capsid protein  Phase extension Abbreviations NCS Non-crystallographic symmetry cryoEM Cryo-electron microscopy D. Nemecek  P. Plevka (&) Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic e-mail: [email protected] E. Boura (&) Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo nam. 2, 166 10 Prague 6, Czech Republic e-mail: [email protected]

1 Introduction The bacteriophage /6 is a lytic dsRNA virus that infects bacteria of Pseudomonas syringae and is the type member of the family Cystoviridae. Cystoviruses share many structural and functional features with other dsRNA viruses, such as a multilayered virion and segmented dsRNA genome, although Cystoviridae. is the only family of dsRNA viruses infecting bacteria. The cystoviruses were for a long time the only dsRNA viruses with well-developed reverse genetics and were therefore used as a model system for other dsRNA viruses, including the human pathogens reoviruses and rotaviruses. Nowadays, plenty of biochemical and genetic data is available that describe replication and maturation of /6 virus [1–5]. The innermost shell (capsid) of the bacteriophage /6 is composed of 120 copies of the P1 protein that are organized into a T = 1 icosahedral lattice of P1 dimers. The two P1 subunits in the icosahedral asymmetric unit adopt different conformations due to their distinct environments in the capsid. In analogy with other dsRNA viruses, the P1 subunits at the fivefold icosahedral axes are denoted P1A, whereas those spanning the twofold and threefold axes are denoted P1B [6]. The capsid is first assembled as an RNA-free precursor (procapsid) that includes several copies of the RNAdependent RNA polymerase (P2), hexameric packaging NTPase (P4), and a minor capsid protein (P7). Subsequently, three single-stranded RNA (ssRNA) segments are specifically packaged into the procapsid in a stepwise manner [4, 7, 8]. The specific order of packaged segments seems to be dependent on the conformation of the icosahedral shell, while packing is driven by the NTPase activity of P4 [9]. Subsequently, the packaged ssRNA strands are replicated to double-stranded RNA (dsRNA) and

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transcribed inside the capsid shell by the viral RNAdependent RNA polymerase [1]. The viral dsRNA genome is thus never exposed to the host cytoplasm, where it would be rapidly degraded. The atomic structure of P1, together with cryoEM reconstructions of the procapsid and nucleocapsid have provided the first atomic model of dsRNA virus maturation that have been discussed elsewhere [10]. Determination of macromolecular structures by macromolecular crystallography is complicated by the impossibility to measure phases of individual reflections. However, the missing phases can be substituted by phases calculated from a model that is structurally similar to the molecules that formed the crystal. Previously, cryoEM maps have been used as phasing models to determine crystal structures of large protein assemblies such as viral shells and the ribosome [11–13]. ˚ resolution cryoEM map and Here we report the use of a 7 A phase extension to determine the crystal structure of the /6 ˚ resolution. The self-rotation P1 major capsid protein at 3.6 A function together with the analysis of asymmetric unit content unambiguously indicated that the P1 crystals were composed of pentamers. Thus electron density map of one P1 pentamer ˚ resolution cryoEM reconstruction of empty /6 cut from 7 A capsid could be used as an initial molecular replacement model. The phases for reflection with resolutions from 7 to ˚ were determined by phase extension. 3.6 A

2 Materials and Methods Plasmid construction, protein expression, purification, crystallization and data collection are described in detail elsewhere [10]. Genes for expression of the full-length P1 and truncated P7 (residues 1–150) proteins were modified to encode a C-terminal 6xHis tag and cloned into the first cassette of the pRSFD vector. The proteins were overexpressed overnight at 20 °C after induction with 0.5 mM isopropyl thiogalactoside (IPTG) in Escherichia coli BL21(DE3) Star cells at optical density (OD) = 0.8. The proteins were purified from the cell extract with affinity chromatography, using Ni–NTA resin (QIAGEN). P1 was further purified with size-exclusion chromatography using a Superdex 200 column (GE Healthcare). Fractions corresponding to the monomer peak were pooled and concentrated to -8 mg/ml, flash-frozen in liquid nitrogen, and stored at -80 °C. For crystallization a mixture of P1:P7 in a 2:1 molar ratio at a total concentration of 2 mg/ml was used. Crystals were grown in hanging drops consisting of a 1:1 mixture of the protein and well solution (100 HEPES, pH = 7.5, 180 mM calcium acetate, 10 mM EDTA, and 39 % PEG 400) at 293 K. The data were collected at the beamline 22-ID, Advanced Photon Source, at 100 K using the MARMOSAIC 300 mm CCD detector. Although the ˚ , they were prone to radiation crystals diffracted to 3.6 A

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damage and the diffraction resolution was decreasing rather rapidly. Therefore, we had to merge data from three crystals ˚ resolution. The quality to obtain a complete dataset at 3.6 A indicators of the resulting dataset were published previously [10] and are not shown here. Data processing, merging and integration was done using the HKL2000 suite [14].

3 Results 3.1 X-ray Structure Determination The diffraction data collected from P1 crystals were indexed and integrated in base centered orthorhombic system using the program HKL2000. The space group was determined to be C2221 by observing that reflections h = 0 k = 0 l = (2n ? 1) had systematically low values. The space group was further verified with program Pointless from the CCP4 package [15]. Matthews coefficient indicated that crystallographic asymmetric unit may contain four to seven P1 molecules (Table 1). The self-rotation function, calculated with the program GLRF [16], showed presence of fivefold and twofold noncrystallographic symmetry (Fig. 1). The axes appeared to be arranged in 52 point group with crystallographic twofold axis congruent with one of the twofold axes on the 52 point group. Thus, the rotation function, together with the Matthew’s coefficient, indicated that there is one pentamer of capsid proteins per crystallographic asymmetric unit. The density map of a P1 pentamer was extracted from the segmented cryoEM map (accession number EMD-2341) of ˚ resolution [17]. Segmentathe /6 whole procapsid at 7-A tion of the cryoEM map had been done in Chimera [18] using the Segment Map function [19] and manual refinement of the over-segmented map. To simplify interpretation of the icosahedral map, segmentation was performed on a subset of asymmetric units that constituted three neighboring fivefold vertices. The EM density was contoured at 4-sigma to visualize characteristic rod-like densities of a-helices.

Table 1 Matthews coefficients for different asymmetric unit compositions Number of molecules in asymmetric unit

Matthews coefficient

Solvent content (in percents)

4

4.61

73.36

5

3.69

66.70

6

3.08

60.04

7

2.64

53.38

8

2.31

46.72

9

2.05

40.06

10

1.85

33.40

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Fig. 1 Rotation functions calculated from phi6 major capsid protein crystal diffraction data. Stereographic plots of (a) two- and (b) five˚ resolution data fold rotation functions were calculated using 5–3.6 A ˚ radius of integration. The plots are contoured starting from and 70 A

0.5 sigma in 0.5 sigma increments. Please note that the relative positioning of the twofold and fivefold axes indicates presence of 522 point group symmetry. Crystallographic twofold axis along a is congruent with one of the twofold axes of the 522 point group

˚ Fig. 2 a Segmented cryoEM map of the /6 procapsid at 7 A resolution. The P1A subunits (blue and green) compose inverted fivefold vertices, whereas the P1B subunits (red and yellow) connect the vertices into an icosahedral shell. Inversion of the P1A subunits into the procapsid interior facilitated segmentation of the P1A and P1B subunits by distinction of intersubunit interfaces in three dimensions [17]. b Extracted map (grey density) of the P1A pentamer that was used for crystal structure determination by molecular replacement.

The final structure of one P1 subunit (red ribbon) is fitted into the map showing close correspondence between the cryoEM map and the crystal structure. Only few helices deviate significantly (arrow) due to interactions with the P1B subunits in the procapsid. c A detailed view at the crystal structure (red ribbon) fitted into the cryoEM map of a part of one P1 subunit. Helices and the b-sheet of the P1 structure fit well into the cryoEM map (Color figure online)

Automatically segmented densities by the Segment Map function were merged manually based on their connectivity and the fivefold symmetry of the vertex. During this procedure, similar features in the two non-symmetry-related subunits became obvious and guided further merging of individual segments. The two conformers of the P1 subunit (P1A and P1B) in the procapsid shell are relatively rotated about 90°, while the P1A subunits are substantially inverted

into the procapsid interior (Fig. 2a). Consequently, the edges of the P1B subunit are clearly separated from the neighboring subunits. Comparison of the structural features in the P1B and P1A subunits then facilitated unambiguous segmentation of the P1A subunits around the fivefold icosahedral axis. Finally, the segmented P1A and P1B subunits were compared to refine the exact boundaries between the subunits in the procapsid shell.

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We generated 11 variants of the P1A pentamer map at different relative magnifications (ranging from 95 to 105 % of the original map with 1 % steps) to be used as models for the molecular replacement, because electron microscopes can have an error in magnification calibration of up to 5 %. Each of the eleven cryoEM maps was placed inside ˚ 9 500 A ˚ 9 500 9 P1 unit cell. Structure factors a 500 A were calculated by Fourier inversion of the electron density maps using the program Sfall of the CCP4 package [15]. The structure factor files were in turn used as molecular replacement models for molecular replacement by the program Phaser [20]. The map scaled to 99 % of the original size performed best and gave good score in the molecular replacement searchs (LLG = 470, TFZ = 22, R-factor = 50.4 %) indicating that the solution was found. Because of the limited resolution of the cryoEM map, the initial phases were useful only for structure factors with ˚ . The phases for higher resolution resolution lower than 7 A reflections were obtained by phase extension that utilized the fivefold non-crystallographic symmetry of the capsid protein pentamers present in the crystal. Orientation of the fivefold non-crystallographic symmetry axis was determined from fivefold self-rotation function. Position of the pentamer was determined by translation search in Phaser. The mask defining the volume of electron density to be averaged was determined based on real space correlation map calculated from the electron density map based on the initial molecular replacement phases and fivefold NCS with the program Coma from the USF package [21]. The initial molecular replacement phases were refined with 15 cycles of real space averaging using the program AVE [22]. In the phase extension procedure, phase information for reflections immediately outside the current resolution ˚ -1 limit was obtained by extending the resolution by (1/b) A followed by five cycles of averaging at each step. This ˚ resolution limit had procedure was repeated until the 3.6 A been reached. Orientation and position of the fivefold noncrystallographic symmetry axis were refined by changing these in small steps and searching for the highest correlation between observed and calculated structure factor amplitudes.

3.2 P1 Structure The biological implications of its structure have been discussed in more detail elsewhere [10]. The P1 monomer has a trapezoidal shape (Fig. 3) with the dimensions *90 ˚ 9 75 A ˚ and with varying thickness of about 14–47 A ˚. A No fold similar to that of P1 was found in DALI search at the time when the structure was solved. Only very recently, a very similar fold has been found in the major capsid protein of the related bacteriophage /8 (Fig. 3) [23]. The

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Fig. 3 Comparison of the P1 protein of bacteriophage /6 (shown in turquoise) and the P1 protein of bacteriophage /8 (shown in magenta, [23]). The overall fold is similar with the largest differences in the loopy regions (arrows) (Color figure online)

fold is mostly a-helical with few short b-sheets. A detailed comparison of P1-/6 and P1-/8 can be found in [24]. Lysine residues 119 at the very tip of the trapezoidal P1 subunits project into the central channel of the P1 pentamer. This channel corresponds to the presumed RNA-translocation channel in the fivefold icosahedral axis of the procapsid. These lysine residues likely interact with phosphate groups of the ssRNA segments of the /6 genome during their packaging into preassembled procapsids and during release of newly transcribed ssRNA segments from mature nucleocapsids. These lysine residues likely interact with phosphate groups of packaged or replicated RNA segments and facilitate their translocation into and from the capsid shell. Interestingly, the conformation of this region of the P1 pentamer is affected by binding of the accessory protein P7 that facilitates RNA packaging [17].

4 Discussion Although the completeness of the data in the highest resolution shell is only 41.6 %, the effective completeness is over 200 % due to the fivefold NCS. The quality of the phases derived by phase-extension was sufficient for initial automated model building using the program Buccaneer [25] (Fig. 2b). Already the first model had about half of the primary sequence assigned and reasonable statistics of Rwork = 30 % and Rfree = 38 %. The model included almost the whole main chain (residues 1-761 out of 769, the C-terminal His6 tag was also disordered). After many rounds of manual rebuilding in Coot and NCS-restrained refinement in Phenix [26], we were able to assign the

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Fig. 4 a Side view of the P1 pentamer. Lysine residues 119 at the tip of each monomer are shown in stick representation and labeled. Each monomer is shown in a different color. b External view at the phi6 procapsid along the fivefold icosahedral axis showing the density of

the P1A pentamer (blue) that was extracted and used in structure determination of the P1 subunit. c Part of the P1 monomer in a 2Fo— Fc map contoured at one sigma (Color figure online)

whole sequence (residues 1–761) and model all sidechains but several flexible Lys and Arg residues located in loop regions. Our final model was refined to exceptionally good value of Rwork = 21.7 % and Rfree = 27.4 % with good stereochemistry. For the fit of P1 into the electron density see also Fig. 4c. We have not found using the sum-of-pairs method as implemented in the DALI server any similarity between the fold of the major capsid proteins of bacteriophage /6 and other type dsRNA viruses that each has a unique fold and organization of the major capsid protein including the bluetongue virus (pdb ID 2BTV) [27], partivirus (pdb ID 3ES5) [28] and picobirnavirus (pdb ID 2VF1) [29]. Although it has been suggested that all major capsid proteins of the dsRNA viruses contain a common core comprised of roughly 150 residues [23], there are at least four different folds that can accommodate an architecture of 120 subunits in the icosahedral shell. This architecture is typical for dsRNA viruses and unique in the realm of virus structures. The dsRNA viruses thus represent an exception from the general paradigm of structural virology, where the subunit fold is typically conserved while the subunits can assemble into icosahedral shells of different icosahedral architectures (T-numbers) and sizes).

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Acknowledgments This research was supported by MarieCurie FP7-PEOPLE-2012-CIG, project number 333916, and by Academy of Sciences Czech Republic (RVO: 61388963). Crystallographic data were collected at the Southeast Regional Collaborative Access Team 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38.

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Using cryoEM reconstruction and phase extension to determine crystal structure of bacteriophage ϕ6 major capsid protein.

Bacteriophage ϕ6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ϕ6 ...
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