Hydrogen-Bonding Networks and RNA Bases Revealed by Cryo Electron Microscopy Suggest a Triggering Mechanism for Calcium Switches

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Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches

Peng Gea,b,c and Z. Hong Zhoua,b,c,1

aDepartment of Microbiology, Immunology, and Molecular Genetics and the bCalifornia NanoSystems Institute, University of California, Los Angeles, CA 90095-7364; and cStructural Computational Biology and Molecular Biophysics Program, Baylor College of Medicine, Houston, TX 77030

Edited* by Juli Feigon, University of California, Los Angeles, CA, and approved April 14, 2011 (received for review December 8, 2010)

Helical assemblies such as filamentous viruses, flagella, and F-actin TMV has a chemical switch that causes the protein to change represent an important category of structures in biology. As the first when the environment changes. CP has several clusters of acidic discovered virus, tobacco mosaic virus (TMV) was at the center amino acids (including a cluster of negative charges formed by an of virus research. Previously, the structure of TMV was solved at aspartic acid residue and a phosphate moiety of the viral RNA) atomic detail by X-ray fiber diffraction but only for its dormant or that are stable outside of cells, where calcium-ion and proton high-calcium-concentration state, not its low-calcium-concentration levels are relatively high, but repel one another in the low-calcium, state, which is relevant to viral assembly and disassembly inside low-proton conditions inside cells. CP mediates assembly and host cells. Here we report a helical reconstruction of TMV in its disassembly through these carboxyl-carboxylate or phosphate- carboxylate pairs that switch states at different calcium and proton calcium-free, metastable assembling state at 3.3 Å resolution by concentrations (11, 13, 14). Such a regulation is common in plant cryo electron microscopy, revealing both protein side chains and viruses. Each of these clusters chelates a calcium ion at high- RNA bases. An atomic model was built de novo showing marked calcium concentration or shares a proton at high-proton concen- differences from the high-calcium, dormant-state structure. Al- tration; and, due to their negative charges, amino acid residues though it could be argued that there might be inaccuracies in the within each of these clusters repulse one another in the absence of latter structure derived from X-ray fiber diffraction, these differen- a calcium ion or proton. Previously, a 2.9 Å resolution structure of ces can be interpreted as conformational changes effected by TMV in high-calcium concentration was solved by X-ray fiber calcium-driven switches, a common regulatory mechanism in plant diffraction (6). In contrast, TMV is relatively poorly understood in viruses. Our comparisons of the structures of the low- and high- its low-calcium state. TMV structures in its calcium-free confor- calcium states indicate that hydrogen bonds formed by Asp116 mation have been determined by cryoEM at 9 Å (15) and 4.7 Å and Arg92 in the place of the calcium ion of the dormant (high- (9). However, the limited resolutions in these structures have calcium) state might trigger allosteric changes in the RNA base- precluded the identification of protein–RNA interactions neces- binding pockets of the coat protein. In turn, the coat protein–RNA sary to establish the mechanism of viral assembly. interactions in our structure favor an adenine-X-guanine (A*G) mo- The high redundancy intrinsic to helical objects makes them tif over the G*A motif of the dormant state, thus offering an expla- good subjects for cryoEM averaging. However, no helical cryoEM nation underlying viral assembly initiation by an AAG motif. structures determined to date have surpassed the 4 Å barrier, thus prohibiting de novo full-atom model building. Examples include the nicotinic acetylcholine receptor (16) and the bacterial flagellar elical assemblies represent a very important class of biological filament (17), structures of which have reached but not surpassed Hstructures, performing various tasks for the proper functions 4 Å resolution. Limitations in both instrumentation and data of life or executing malicious functions in disease. For example, processing may have contributed to this resolution barrier. On the protein helical assemblies such as cytoskeletal networks and one hand, magnification variability due to nonparallel illumination muscle fibers are essential components in our bodies (1, 2). Other fl and phase errors due to beam tilt can severely limit the resolution helical assemblies include bacterial agella and secretion systems. of the cryoEM structures obtained. Recently, techniques such as Helical viruses, such as tobacco mosaic virus (TMV), account for parallel illumination and coma-free alignment have been used in a major fraction of the virus kingdom. Such helical structures, cryoEM (18, 19), eliminating many of the resolution barriers im- bearing a special kind of 2D periodicity and potential flexibility, are fi posed by previous imaging systems. On the other hand, previous dif cult for structural determination to atomic resolution by con- helical reconstruction methods have demanded “perfect” helical ventional methods including X-ray crystallography and NMR. symmetry for higher-resolution reconstructions, a condition that Thus, the molecular mechanisms of action in many of these sys- cannot be met by helical assemblies in reality. The iterative helical tems are not understood to atomic detail. Even for TMV, the first real-space reconstruction method (IHRSR) (20) has made it MICROBIOLOGY virus ever isolated and extensively studied (3) by both X-ray dif- – possible to deal with such imperfections. fraction (e.g., 4 8) and cryo electron microscopy (cryoEM) (9, 10), Here we take advantage of these latest technical developments we still do not know its structure at atomic detail in a state that is and further improve the helical reconstruction method. By doing relevant to its assembly and disassembly processes, and the un- so, we have determined the structure of TMV in its metastable derlying mechanisms of these processes remain unknown. calcium-free state at 3.3 Å resolution by cryoEM helical re- The viral RNA of TMV is infectious by itself, but RNA on its construction. Our structure reveals the mechanisms by which the own is very unstable. The addition of a 17-kDa coat protein (CP) around the RNA protects the RNA from degrading and makes TMV very stable. The assembly of a rod-shaped virion begins at a two-turn helical CP complex, called the 20S aggregate (11), which Author contributions: P.G. and Z.H.Z. designed research; P.G. performed research; P.G. recognizes the initiation motif. This motif contains an AAG repeat and Z.H.Z. analyzed data; and P.G. and Z.H.Z. wrote the paper. (11), with the last G being the most conserved (12). Each mature The authors declare no conflict of interest. TMV is composed of 2,130 CP molecules and a 6-kb linear posi- *This Direct Submission article had a prearranged editor. tive-strand RNA, organized in a helical rod of 3,000 Å in length Data deposition: The cryoEM density map and the atomic model have been deposited and 180 Å in diameter. With the protection of CP, TMV can in the EMDB and Protein Data Bank (ID codes EMD-5185 and 3J06, respectively). survive for years in cigars and cigarettes made from infected leaves. 1To whom correspondence should be addressed. E-mail: [email protected]. The protein coat poses a problem, however, because CP must This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. be removed once the virus gets inside a cell. As for many viruses, 1073/pnas.1018104108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1018104108 PNAS | June 7, 2011 | vol. 108 | no. 23 | 9637–9642 Downloaded by guest on September 27, 2021 TMV coat protein swaps base favorability (G*A to A*G) in re- S2A). In our strategy, projection generation and matching are only sponse to the removal of calcium ions from its calcium switches. performed at azimuthal angle locations 0, 1θ − 1δ,2θ − 2δ...until Our structure also explains why both assembly and disassembly 360° is reached, in which (θ − δ)° is the size of an actual angular step can happen with the same components in the same environment. (which is the angular difference between two neighboring projections within the same altitude) and where θ is an arbitrarily chosen Results angle that is much larger than the desired angular interval δ.(Due Determining Helical Structure to Atomic Resolution. As detailed in to space limitations, a brief description is provided below. For more Methods, we optimized the acquisition of high-resolution images detailed guidelines, see SI Discussion.) in three aspects. First, we used a thoroughly cleaned and exten- We first find the upper limit of δ based on the targeted reso- sively “baked” holey carbon grid as well as a small beam size to lution and the Crowther formula (21). For the case of TMV, δ has minimize specimen charging. Second, we used parallel illumination to be less than 1° to achieve 3.3 Å resolution; it also has to be an ϕ to eliminate magnification differences across images at different integral fraction of the angular span of the asymmetric unit, (in ϕ defocus settings. Third, we minimized beam tilt to reduce phase the case of TMV, = 22.04°), for the vernier strategy to work. δ ϕ ϕ errors. Electron micrographs of frozen hydrated TMV in the meta- Therefore, the available choices of for TMV here are /22, /23, ϕ stable calcium-free state show characteristic striations perpendic- /24, and so forth. Then we experiment on different combinations θ δ θ ular to the helical axes (Fig. 1A). These particles were boxed into of and that satisfy the following three relationships: First, is A an integral fraction or an integral multiplicity of ϕ (i.e., ϕ =mθ); segments (see box in Fig. 1 ) which were subjected to reference- δ θ θ δ × free 2D classification to obtain class averages. The incoherent av- second, equals an integral fraction of ( =n ); and third, n m is an integer. The nominal angular locations (0, 1θ − 1δ,2θ − erage of Fourier transforms of these class averages shows layer δ... lines beyond a spacial frequency of 1/(3.2 Å) (Fig. S1A), demon- 2 ), in turn, fall into different asymmetric units at effective angular positions 0, (n − 1 mod mn)δ, (2n − 2 mod mn)δ, (3n − 3 strating the resolution limit of the images beyond 3.2 Å. mod δ... Reconstructing helical objects with IHRSR (20) to high reso- mn) relative to the beginnings of each asymmetric unit i when helical symmetry is considered (Fig. S2B). After calculating lution requires meeting the following two conditions: ( ) To ob- θ δ tain a TMV structure at 3.3 Å resolution, the angular step size of these numbers, we would know whether a certain ( , ) pair passes the last quality control: whether the effective angular positions the projections has to go down to 1° as predicted by the Crowther δ δ... − δ formula (21); and (ii) to use single-particle methods as a re- cover all of the 0, ,2 (mn 1) positions in the asymmetric construction engine, the particles’ orientations should cover the unit and whether these effective angular positions are evenly dis- azimuthal (rotation about the helical axis) range of 360°, as tributed within the asymmetric unit (the number of nominal pro- implemented in the original IHRSR (20). Consequently, the total jection locations can be more than the effective positions; in this number of projections required, considering the out-of-plane tilt, case, we would like the effective positions of the extra projections could rise up to several thousand and the resulting computation to be evenly distributed in the asymmetric unit). If it does not, we try a different pair of θ and δ. load of data processing could become prohibitive. Considering fi θ helical symmetry, we only need to sample within the helical Following these strategies, one would nd at least one pair of and δ that satisfy the criteria described above (see SI Discussion asymmetric unit. Therefore, we add to IHRSR a vernier angular B sampling strategy based on the principle of the vernier caliper (Fig. for detailed guidelines). As illustrated in Fig. S2 , the effective azimuthal positions of the nominal angular locations (0, 1θ − 1δ, 2θ − 2δ...) are, after sorting, 0, δ,2δ...(mn − 1)δ in the asymmetric unit. In this way, we fulfill both requirements (i)and(ii) while reducing computational cost by n-fold (in the case of TMV, 11-fold). We note that this method could in some cases have two minor imperfections: (i) If for a desirable δ we cannot find m, n, and θ so that mn(θ − δ) = 360° then we have to bear with mn(θ − δ) < 360°, and some locations in the asymmetric unit will have to be visited more than once; and (ii) the method works best with a helical pitch (rise per turn) less than or comparable to half the nonoverlapping part of a segment (box size minus overlapping, i.e., new data represented by a box) (SI Discussion). For TMV, the helical pitch is 22.9 Å. The box size of each image is 512 pixels (445 Å). We used a 90% overlapping schema, and thus the nonoverlapping part of a segment (44.5 Å) is very close to twice its helical pitch (45.8 Å). These imperfections, although undesirable, did not prohibit us from achieving atomic resolution. In this study, the vernier parameters θ and δ we used are 540/49 = 11.02° and 45/49 = 0.918°, respectively (θ =12δ); the nominal azimuth step size of projections is θ − δ = 10.1°; θ equals half the angular span of an asymmetric unit (ϕ =2θ). The resolution of our final map is 3.3 Å based on the 0.5 criterion in the Fourier shell correction coefficient between maps from two randomly divided half-datasets from the full dataset (Fig. S1C). The resolving power of our cryoEM map can also be judged from the quality of the densities of the amino acid side chains, RNA bases, and backbone carbonyls (Figs. 2 and 3 and Fig. S3). To judge the resolving power of the map based on geometrical Fig. 1. CryoEM reconstruction of TMV at 3.3 Å resolution. (A) CryoEM mi- fi crograph showing natural striations of the TMV rods embedded in vitreous criteria, we nd that several pairs of density peaks 3.3 Å away ice. (Scale bar, 600 Å.) The image was denoised by applying a wiener filter, (including the densities for the base regions of base 1/base 3 and fi fi the densities for the connecting phosphate/sugar groups along the a median lter, and a low-pass lter to enhance the contrast. (B) Central slice A–C from the final 3D reconstructed map. (C) Overview of the helical density RNA backbone) are well-separated (Fig. 2 ). We also com- map. (D) A single turn of 16 subunits of capsid protein (ribbons in rainbow pare our density map side by side with X-ray structures at similar colors) and bound RNA (atomic model at low radius). (E) Ribbon diagrams of resolutions, such as the human hepatitis B virus core at 3.3 Å (22). one CP and its three bound RNA bases superimposed with the cryoEM The side-chain features revealed in our map are comparable to density map. See also Movie S1. LR, RR, LS, RS: left radial, right radial, left those in these X-ray structures at similar resolutions (Fig. 2 D–F slewed, and right slewed α-helices, respectively. and Fig. S3).

9638 | www.pnas.org/cgi/doi/10.1073/pnas.1018104108 Ge and Zhou Downloaded by guest on September 27, 2021 Overall Structure. Our reconstructed TMV structure (Fig. 1 B–E) cepted, we cannot completely rule out the possibility that some of has the same overall shape as that of the high-calcium state solved the differences between the cryoEM and X-ray structures might by X-ray fiber diffraction (6). The calculated helicity of our struc- be due to inaccuracies in structures derived from fiber diffrac- ture is 16.338 subunits per turn, or 49.013 subunits per three turns, tion data. which is slightly different from those used in previous studies (49.02 Of special note is the switch formed by Asp116 and RNA in refs. 6 and 9, and 49.019 in ref. 23). Each asymmetric unit of TMV phosphate (Fig. 4 C and D). Structural comparison of the high- contains one CP and three RNA bases (Fig. 1E). Each CP consists calcium and calcium-free conformations reveals that the removal of four major helices, two minor helices, and one tiny β-sheet (Fig. of Ca2+ triggers conformational change in CP. Upon removal of 2E). These secondary-structure elements are joined by loops, one of the calcium ion from the center of the switch, RNA moves away which runs radially and binds RNA (RNA-binding loop) (Fig. 1E). from Asp116 due to electrostatic repulsion. Asp116, now isolated, These loops are well-resolved in our cryoEM structure, unlike the attracts Arg92 from an upper turn to form two hydrogen bonds, previous lower-resolution cryoEM structure (9). moving the RNA-binding loop 5 Å closer to Asp116 (Fig. 4 C and Based on this map, we built a full-atom model de novo (Meth- D). Another arginine (Arg90) on the same RNA-binding loop ods). In each CP subunit, 155 ordered residues (Ser3–Ala157) and moves with the loop and fills the original position of Arg92 (Fig. 4 3 bases are resolved. Using side-chain densities resolved in our C and D). Consequently, the side chains of these two arginines map and the counting criterion as described previously (24), we sandwich the phosphate backbone of RNA by hydrogen bonds identified in each CP 113 fully resolved amino acids (73%), 24 (Fig. 4D). In this conformation, Arg90 and Arg92 neutralize the partially resolved residues (16%), 11 ambiguous residues (7%), negative charges of the backbone phosphate of base 2 and the and 6 glycines (4%). Notably, even most of the “ambiguous” and carboxylate group of Asp116. Also moving with the RNA-binding glycine residues have defined Cα positions. Carbonyl groups are at loop is Glu95, which forms one hydrogen bond with Arg112 using times (>15%) visible in the density as protrusions (Fig. 3). The its backbone oxygen and another hydrogen bond with the sugar RNA densities are also well-resolved. Phosphate groups sharply moiety of base 3 using its side chain (Fig. 4F). Therefore, the stand out at higher contour levels. The densities for the base calcium-free, assembling conformation of TMV fashions an al- moieties are in pie shapes similar to those of tryptophan residues. ternative network of hydrogen bonds among CPs over two helical turns and their integral RNA, strengthening the affinities between Low-Calcium State of the Calcium Switches. There are three an upper CP and RNA and between CPs across two helical turns switches (each switch composed of a juxtaposed pair of negative at the price of the affinity between RNA and a lower CP (Fig. 4D), charges) per CP in the TMV virion; two of them bind calcium. Of compared with the high-calcium conformation. Counterintui- the two, one is formed by Glu106 and Glu95; Glu97 and Asp109 tively, the opening of this calcium switch does not contribute to the in their vicinity were found to also participate in this switch (25) disassembly of the virion but instead to axial stability. (Fig. S4, blue circle). Another is formed by the carboxylate of The other calcium switch, located closer to the helical axis Asp116 and the phosphate moiety of base 2 of the bound RNA (Fig. S4, blue circle), has been described previously (9). The (Fig. S4, red circle). The structure of these switches in their high- opening of this second switch results in lower affinity between calcium states has been shown by X-ray fiber diffraction (6). adjacent CP subunits in the lateral direction. Unlike the closed state in the high-calcium structure (e.g., Fig. In sum, the net effect of losing calcium ions in the calcium 4C), these two switches are both in the open (repelled) state in switches is threefold: The lateral interaction between CPs is our cryoEM structure (e.g., Fig. 4D). Despite the fact that the considerably weakened; the axial interaction between CPs is high-calcium structure from X-ray fiber diffraction is widely ac- strengthened; and the affinity between CP and bound RNA is decreased. Such effects have changed the tightly bound helical assembly into a loosely bound “beads on a string” structure that is held together by encircling (but not as strongly binding to) the viral RNA, making possible the cotranslational stripping of CP by ribosomes during disassembly (6, 26–28).

Changes in CP–RNA Base Hydrogen-Bonding Networks Triggered by Low-Calcium Concentration. Our structure of the low-calcium state differs in many ways from the previous structure of the high- calcium state. Therefore, when a TMV in its high-calcium, dormant state encounters the very low calcium concentration environment of the cytoplasm of a plant cell, CP undergoes marked conformational changes. These changes result in the breakage and rear- rangement of several hydrogen bonds between RNA and CPs. Among the binding partners of base 1 in the high-calcium state, Arg122 turns away and no longer forms a hydrogen bond with base 1. Instead, it forms a salt bridge with Asp88. The access of Asp115 MICROBIOLOGY to the N2 of a guanine at base 1 is sterically occluded by the side chain of Glu36. The side chain of Thr89, which used to form a hydrogen bond with base 1, now forms a hydrogen bond with base 3(Fig.5A and B). Thus, the hydrogen-bond network that has made guanine favorable for base 1 in the high-calcium state no longer exists. A new hydrogen-bond network that favors both adenine and guanine with a slight tendency toward adenine now forms around Fig. 2. Resolution assessment of the 3.3 Å cryoEM map. (A–C) Represen- base 1: The backbone oxygen of Gly85 selectively forms a hydrogen tative structural features resolved beyond 3.3 Å resolution. Atomic models A are shown as sticks and cryoEM densities are shown as wire frames. In B,the bond with the unique N6 of an adenine (Fig. 5 ); the backbone oxygen of Asp88 selectively forms a hydrogen bond with the unique higher-density regions are displayed in navy color, showing that sugar and B phosphate groups 3.3 Å apart are well-resolved. (A and C) The two stacked N2 of a guanine (Fig. 5 ); Asp115, when protonated, can form base groups of the RNA (A) and the side chains of the two arginines that are a hydrogen bond with N3 of any purine, although adenine is more stacked with their π-bonds (C) are 3.3 Å apart. (D–F) Representative regions favorable because it is more basic. The last aspect explains the slight of the map ﬁtted with their atomic model, showing the quality of the map. favorability toward adenine for base 1 at pH 5.4, as reported in an (D and F) Two hydrophobic regions showing the quality of the side-chain earlier study (29). densities. (E) The sole small β-sheet region of CP, showing the separation of On the other hand, base 3 now favors guanine. The backbone the strands. See stereo views of D–F in Fig. S2. oxygen of Thr89 now turns and its hydrogen bond with base 1 in the

Ge and Zhou PNAS | June 7, 2011 | vol. 108 | no. 23 | 9639 Downloaded by guest on September 27, 2021 Ordering of the Inner Loop of the Coat Protein Through Hydrogen Bonding. It was thought that the ordering of the inner loop plays a critical role in controlling viral assembly (11). In the cryoEM structure, although the inner loop does not contribute to lateral affinity, it is in a highly ordered conformation and therefore contributes to the formation of a longer helical aggregate. Due to the opening of the low-radius calcium switch (blue circle in Fig. S4), Glu106 and the turn it belongs to in the X-ray fiber diffraction structure are now allowed to extend the left radial (LR) (6) helix. On the other side, the aforementioned formation of hydrogen bonds between Glu95 and Arg112/base 3 has directed the segment that was originally a loop (amino acids 91–101) to fold into an ordered structure of multiple turns. Two acidic residues, Glu106 and Glu97, are now within hydrogen-bonding distance in the same Fig. 3. Carbonyl densities. Representative areas of the density map that subunit. When protonated, they can form a hydrogen bond. This match with the shape of corresponding backbone carbonyl groups (arrows). hydrogen bond also contributes to the ordering of the inner loop region. The remaining residues (three) that do not assume any high-calcium state is broken. As a consequence, its side-chain ox- secondary structure (helices, sheets, or turns) are of minimal ygen now forms a hydrogen bond with base 3 (Fig. 5D). This hy- length and are fully stretched at the lowest radius. drogen bond can be formed for all bases. If this base is a guanine, an additional hydrogen bond may form between its N2 and Gln39 Discussion (Fig. 5D). Thus, guanine is more favorable at this base position. Through the determination of the calcium-free TMV structure to For base 2, the size of the density is too small for a purine; 3.3 Å resolution, we demonstrate that 3D structures of helical therefore, we model a uracil base at this location (Fig. 5C). O4 of objects can be determined by cryoEM to atomic resolution. Full this uracil forms a hydrogen bond with Arg46 (Fig. 5C). This atomic models by the cryoEM method were previously only ach- hydrogen bond can also be formed with a guanine. A water ieved for single particles with icosahedral symmetry (18, 19). molecule mediates a hydrogen-bonding network between Arg90, Reaching such resolution for helical objects opens the door for C atomic-resolution structural determinations of important systems Thr42, and N3 of this uracil (Fig. 5 ). This hydrogen bond can such as amyloid fibers, flagellar filaments, and actin. At a resolu- be formed for all bases. It was suggested that this base favors tion of around 3 Å, main chains can be unambiguously traced and adenine (29); however, in our experiment, we were unable to side-chain densities are readily visible. The basic and aromatic capture the conformation of an adenine bound in base 2, possibly residues seem to have been best resolved. Nonaromatic hydro- due to the relatively low abundance of adenine at this base po- phobic residues are well-resolved too. It is noteworthy that the sition in the whole viral genome. side chains for about 70% of the acidic residues are absent in our The above observations about the changed hydrogen-bonding structure, a phenomenon that may be explained by the lower network at low-calcium concentration on the three base slots are electron dose tolerance of the negatively charged residues. consistent with the notion that an AAG-repeating motif is nec- Low-calcium concentration and high pH were long thought to essary to initiate the encapsidation process (12). be the trigger for TMV virion uncoating (6, 13). Higher calcium

Fig. 4. Conformation changes in the vicinity of the higher-radius calcium switch. The blue and red ribbon models in this ﬁgure and Fig. 5 distinguish two neighboring subunits of CP. (A) Two adjacent subunits are shown in ribbon diagram. The red circle marks the position of the higher-radius calcium switch. (B) Cry- oEM density map around the calcium switch superimposed with its atomic model. For clarity, the RNA base moiety of base 1 is not shown. (C and D) Con- formational changes around this calcium switch between the high-calcium state (C) (from Protein Data Bank ID code 2TMV) and the low-calcium state (D, shown in stereo). Upon losing the bound calcium ion, the phosphate group of base 2 and the carboxylate group of Asp116 repulse each other with their negative charges. Arg92 is attracted into the pocket toward Asp116, dragging the CP backbone with it, thus plac- ing Arg90 in the position occupied by Arg92 in the high-calcium state. As result, Arg90 in the calcium-free conformation interacts with the phosphate group of base 2. Arg92 now makes one hydrogen bond with the phosphate group of base 2 and two hydrogen bonds with Asp116. (E and F) Ordering of the inner loop structure in the low-calcium state (F, shown in stereo) compared with that of the high-calcium state (E). This ordering results in the increased length of the LR helix (arrows) by two and a half turns. For clarity, RNA base moieties are not shown in these two panels. In the calcium-free conformation, Glu95 makes hydrogen bonds with Arg112 and base 3. The two negatively charged residues, Glu97 and Glu106, are only 3 Å apart, indicating that they likely share one proton between them.

9640 | www.pnas.org/cgi/doi/10.1073/pnas.1018104108 Ge and Zhou Downloaded by guest on September 27, 2021 concentration protects TMV from disassembly by possible mod- the Glu95–base 3, Asp116–Arg92, and Arg112–Glu95 hydrogen erately high pH during its dissemination. The lower calcium bonds. Because an oligomer of CP contains multiple subunits, its concentration and the higher pH in the plant cell relative to the affinity to the existing virion is much higher than that of a single extracellular environment allow the first several subunits of CP to CP. Therefore, addition of oligomers of CP outperforms the si- fall off so that ribosome-mediated “cotranslational uncoating” can multaneous uncoating of CP in a “lose few, get many” scenario, occur, due to the low affinity between CP and 5′ RNA in which resulting in the assembly of new virions. In contrast, simultaneous guanines are few (6, 26–28). However, assembly and disassembly uncoating of CP at the 3′ end was never observed, suggesting that of TMV take place in the same plant cell; the same environmental the assembly of CP at the 3′ end progresses continuously toward conditions must accommodate the two seemingly opposite pro- the terminus of the RNA. cesses. As one possible explanation, assembly and disassembly In the fiber diffraction structure it was concluded that the RNA may be switched by the concentration of free CP oligomers. In the binding site specifically favors a G*A motif. However, the RNA infection stage there are few virions in the cell; free CPs are scarce. binding site revealed in our structure favors an A*G motif instead. ′ The 5 RNA can be simultaneously uncoated due to the lower Both motifs match with the AAG motif observed as the initiation fi lateral intersubunit af nity between CPs in the absence of calcium. motif (12) (the G*A motif can be converted to *AG by applying On the other hand, in the viral production stage, the CP oligomers ′ a frameshift operation). Because our structure represents the add to the 5 RNA to elongate the virion (30). Because the inner conformation in a low-calcium-concentration environment, which loop of CP takes an ordered conformation upon RNA binding, the is the same as that which the virus encounters during assembly, the assembly of a longer helix is promoted (11). The newly added A*G motif revealed in our structure now offers direct evidence as oligomer and the existing part of the virion have sufficient axial fi to why the AAG motif initiates the encapsidation process (12). af nity between each other even in the absence of calcium due In contrast to the guanines at the third position in the above- to the formation of the above-mentioned arginine sandwich and described A*G motif in the initiation sequence, adenine is by far the most abundant base at base 3 in the whole viral genome. Recall that based on our structure (calcium-free conformation), guanine at position 3 gets one more hydrogen bond than adenine would at the same position. However, in the presence of calcium, adenine at base 3 enjoys a higher binding affinity than other bases (6). Therefore, the high frequency of adenine at base 3 gives rise to more stable interactions between CP and RNA in a high-calcium environment, but not in a low-calcium environment where the virus assembles. These switchable levels of interactions controlled by the calcium-driven switch might be relevant for the optimal functions of TMV virions in both stages of their life cycle: assembly and spread. The virus uses the strong interaction in the low-calcium environment to precisely initiate assembly and the alternative strong interaction in the high-calcium environment for a more stable virion during spreading. The high frequency of adenine at base 3 also helps to prevent assembly initiation from occurring at incorrect positions of the viral genome. Of course, at the beginning, when a virus assembles at low-calcium concentration in plant cells, the adenines that are rich at base 3 would not have known that they will be favorably bound by CP later on in a high-calcium environment. They gain stronger binding with CPs upon exposure to the high-calcium environment when the progeny virus leaves its original cellular environment, thus leading to more stable virions that are optimal for dissemination. Methods Electron Microscopy. TMV sample (vulgare strain in 20 mM phosphate buffer) was prepared by extraction from infected tissue in the presence of EDTA, followed by differential centrifugation steps. The resulting sample was diluted to optimal concentration with Tris-buffered saline (10 mM Tris, 130 mM NaCl, pH 7.4). Before sample vitrification, carbon-coated Quantifoil R1.3/1.2 copper grids (1.4/1 μm actual hole size) were first cleaned by submerging them in

ethylene dichloride for 1 wk to thoroughly remove noncarbon materials from MICROBIOLOGY the grids. We then “baked” these grids by illuminating them overnight under a 100-keV electron beam under a transmission electron microscope. An aliquot of TMV sample was applied to a ”baked“ grid, blotted, and vitrified using an FEI Vitrobot Mark IV freezer. The vitrification parameters were as follows: 22 °C, 100% humidity, 2 μL sample, 10 s wait time, 1–2 s blot time, 1–2 s drain time, blot force 1. A single frozen grid, cooled at liquid-nitrogen temperature, Fig. 5. Environments of the three bases in the low-calcium state shown in was used for all of the 750 micrographs recorded by an FEI Titan Krios elec- stereo. (A and B) Environment of base 1: the cases for adenine (A) and tron microscope operated at 300 kV in a single 7-d session. The microscope guanine (B). The backbone oxygens of Gly85 and Asp88 make a hydrogen was operated with parallel illumination after careful coma-free alignment bond with N6 of adenine and N2 of guanine, respectively. When protonated, to minimize unintentional beam tilt. Images were recorded on Kodak SO-163 Asp115 can form a hydrogen bond with N3 of this purine (blue dashed lines). film with a beam diameter of 1.5 μm at 75,000× nominal magnification. 2 (C) Environment of base 2. As observed in our structure, the size of the The election dosage was 25 electrons per Å . Micrographs were digitized on density of this base fits a pyrimidine. If it is a uracil, its O4 forms a hydrogen Nikon 9000ED scanners at 6.35-μm step size, giving a final nominal step size of bond with Arg46; its N3 participates in the hydrogen-bond network medi- 0.847 Å on the specimen. The actual step size was calibrated to be 0.87 Å ated by a water molecule. (D) Environment of base 3. Thr89 forms a hydro- according to the previously determined axial rise per subunit of TMV (1.41 Å) gen bond with any base in this position with its side-chain oxygen, either as (SI Discussion); thus the calibrated magnification is ∼73,000×. This calibration a donor or an acceptor. Base 3 favors guanine because only guanine gets an is an approximation, because the number 1.41 is also an approximation and extra hydrogen bond with Gln39. the axial rise per subunit may change due to the conformational change be-

Ge and Zhou PNAS | June 7, 2011 | vol. 108 | no. 23 | 9641 Downloaded by guest on September 27, 2021 tween the high- and low-calcium states. However, this calibration agrees well We used 61,708 particle segments in our refinement process. Each segment with calibrations of the same microscope based on other methods. has a length of 512 pixels, or 445 Å. Although a recent paper recommends a large overlap between neighboring segments for objects with small rise Helical Reconstruction and Refinement. Particles on the micrographs were per subunit (33), we initially chose an overlapping level of 90% as previously selected manually by the helixboxer program in the EMAN package (31) and used (2, 9, 32) (SI Discussion). Therefore, each particle segment has 10% (or subsequently boxed out automatically with a custom-designed program 51 pixels) of fresh data, which represents 31 asymmetric units. All these helixbatchboxer that is deposited in the EMAN repository. This program also segments represent about 1.9 million asymmetric units, or 31,882 icosahe- cut the helical particles into segments using user-selected overlapping dral particle equivalents (compare with 20,473 in ref. 18). Bad segments schema. Reference-free 2D classification was done with the EMAN refine2d.py were excluded based on lack of mutual similarity, and about 43,000 seg- module (Results). The starting model was generated in a similar fashion as ments (22,000 icosahedral particle equivalents; compare with 18,646 in ref. fi previously (32). The 3D structure was re ned iteratively as follows for 10 18) were included in the final reconstruction. iterations. For every iteration, the current best structure is used as a reference to obtain a refined structure using the EMAN refine module for one single Atomic Modeling. The atomic model of the TMV CP and bound RNA was round; this refined structure is used to refine the helical parameters by the modeled first in O (34) and then fine-tuned for optimal geometry and Ram- hsearch_lorentz module of IHRSR (20); the newly determined helical param- achandran plot in Coot (35). The resulting model was refined with the CNS eters are used to generate a new structure by helicizing the EMAN re- package (36) as described in our previous study (18) with the following construction with the himpose module of IHRSR (20). We used the vernier improvements. First, the minimization target was the vector residue (“vector” method to guide projection generation in the EMAN refinement (Results). in CNS) instead of the maximum-likelihood amplitude target (“mlf” in CNS), so For the himpose program, we made three modifications that are critical for reaching atomic resolutions. First, we modified it to use the central 1/5 segment that the experimental phase data were also included in addition to the am- of the volume for averaging, instead of the 2/3 that is hard-coded in the original plitude data. Second, to optimize molecular interfaces, we included one IHRSR program. Second, we introduced a new feature so that the helicized map central asymmetric unit (one CP and three RNA bases) and six surrounding fi will not shift or rotate in relation to the starting model as the original program duplicate copies of this asymmetric unit in the re nement. The seven asym- does. Third, we implemented an oversampling scheme into himpose to mini- metric units are restrained by noncrystallographic symmetry according to the mize interpolation errors introduced during averaging and back-and-forth helical symmetry of the virion. The resolution limit was set to 3.1 Å during this fi fi conversions between cylindrical and Cartesian coordinate systems. re nement. The nal R factor accomplished is 25.5% (Rfree 25.6%). Upon reaching convergence of the above iterative refinement, we switched to a refinement method similar to that described previously (9), ACKNOWLEDGMENTS. We thank Ruben Diaz-Avalos, who originally pre- except that we generated our 3D reconstruction by merging class averages pared the TMV sample, which was kindly provided by Bridget Carragher and (as in EMAN) instead of individual particles. The reconstructed volume was Ronald Milligan as a gift. We thank Lei Jin for assistance in initial modeling, helicized in real space with the IHRSR himpose program. An initial atomic Edward Egelman for the source code of IHRSR programs, Hongrong Liu for micrograph development, and Connie Huang, Jennifer Wong, and Justin Chen model was built from the resulting density as described below, and the for micrograph digitization. This work was supported in part by National Fourier amplitude of the resulting density map was rescaled by a one- Institute of Health Grants GM071940 and AI069015 (to Z.H.Z.). We acknowl- dimensional structure factor (24) which was derived from our initial atomic edge the use of the cryoEM facility at the Electron Imaging Center for model. The map was further sharpened by deconvolution of an envelope NanoMachines supported by the National Institutes of Health (Grant function with a 60 Å2 B factor. 1S10RR23057 to Z.H.Z.).

1. Murakami K, et al. (2010) Structural basis for actin assembly, activation of ATP 20. Egelman EH (2007) The iterative helical real space reconstruction method: hydrolysis, and delayed phosphate release. Cell 143:275–287. Surmounting the problems posed by real polymers. J Struct Biol 157:83–94. 2. Fujii T, Iwane AH, Yanagida T, Namba K (2010) Direct visualization of secondary 21. Crowther RA (1971) Procedures for three-dimensional reconstruction of spherical structures of F-actin by electron cryomicroscopy. Nature 467:724–728. viruses by Fourier synthesis from electron micrographs. Philos Trans R Soc Lond B Biol 3. Klug A (1999) The tobacco mosaic virus particle: Structure and assembly. Philos Trans R Sci 261:221–230. – Soc Lond B Biol Sci 354:531 535. 22. Wynne SA, Crowther RA, Leslie AG (1999) The crystal structure of the human hepatitis 4. Stubbs G, Warren S, Holmes K (1977) Structure of RNA and RNA binding site in B virus capsid. Mol Cell 3:771–780. tobacco mosaic virus from 4-Å map calculated from X-ray fibre diagrams. Nature 267: 23. Stubbs G, Makowski L (1982) Coordinated use of isomorphous replacement and layer- 216–221. line splitting in the phasing of fiber diffraction data. Acta Crystallogr A 38:417–425. 5. Namba K, Stubbs G (1986) Structure of tobacco mosaic virus at 3.6 Å resolution: 24. Zhang J, et al. (2010) Mechanism of folding chamber closure in a group II chaperonin. Implications for assembly. Science 231:1401–1406. Nature 463:379–383. 6. Namba K, Pattanayek R, Stubbs G (1989) Visualization of protein-nucleic acid 25. Lu B, Stubbs G, Culver JN (1996) Carboxylate interactions involved in the disassembly interactions in a virus. Refined structure of intact tobacco mosaic virus at 2.9 Å – resolution by X-ray fiber diffraction. J Mol Biol 208:307–325. of tobacco mosaic tobamovirus. Virology 225:11 20. ′ 7. Pattanayek R, Elrod M, Stubbs G (1992) Characterization of a putative calcium- 26. Mundry KW, et al. (1991) Complete uncoating of the 5 leader sequence of tobacco binding site in tobacco mosaic virus. Proteins 12:128–132. mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus 8. Pattanayek R, Stubbs G (1992) Structure of the U2 strain of tobacco mosaic virus disassembly in vitro. J Gen Virol 72:769–777. refined at 3.5 Å resolution using X-ray fiber diffraction. J Mol Biol 228:516–528. 27. Shaw JG, Plaskitt KA, Wilson TM (1986) Evidence that tobacco mosaic virus particles 9. Sachse C, et al. (2007) High-resolution electron microscopy of helical specimens: A disassemble contranslationally in vivo. Virology 148:326–336. fresh look at tobacco mosaic virus. J Mol Biol 371:812–835. 28. Wilson TM (1984) Cotranslational disassembly of tobacco mosaic virus in vitro. 10. Clare DK, Orlova EV (2010) 4.6 Å cryo-EM reconstruction of tobacco mosaic virus from Virology 137:255–265. images recorded at 300 keV on a 4k × 4k CCD camera. J Struct Biol 171:303–308. 29. Schuster TM, et al. (1980) Studies on the mechanism of assembly of tobacco mosaic 11. Caspar DL, Namba K (1990) Switching in the self-assembly of tobacco mosaic virus. virus. Biophys J 32:313–329. Adv Biophys 26:157–185. 30. Butler PJ (1984) The current picture of the structure and assembly of tobacco mosaic 12. Zimmern D (1977) The nucleotide sequence at the origin for assembly on tobacco virus. J Gen Virol 65:253–279. – mosaic virus RNA. Cell 11:463 482. 31. Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: Semiautomated software for high- 13. Wang H, Planchart A, Stubbs G (1998) Caspar carboxylates: The structural basis of resolution single-particle reconstructions. J Struct Biol 128:82–97. – tobamovirus disassembly. Biophys J 74:633 638. 32. Ge P, et al. (2010) Cryo-EM model of the bullet-shaped vesicular stomatitis virus. 14. Stubbs G (1999) Tobacco mosaic virus particle structure and the initiation of Science 327:689–693. disassembly. Philos Trans R Soc Lond B Biol Sci 354:551–557. 33. Egelman EH (2010) Reconstruction of helical filaments and tubes. Methods Enzymol 15. Jeng TW, Crowther RA, Stubbs G, Chiu W (1989) Visualization of α-helices in tobacco 482:167–183. mosaic virus by cryo-electron microscopy. J Mol Biol 205:251–257. 34. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building 16. Miyazawa A, Fujiyoshi Y, Unwin N (2003) Structure and gating mechanism of the protein models in electron density maps and the location of errors in these models. acetylcholine receptor pore. Nature 423:949–955. – 17. Yonekura K, Maki-Yonekura S, Namba K (2003) Complete atomic model of the Acta Crystallogr A 47:110 119. bacterial flagellar filament by electron cryomicroscopy. Nature 424:643–650. 35. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. 18. Zhang X, Jin L, Fang Q, Hui WH, Zhou ZH (2010) 3.3 Å cryo-EM structure of Acta Crystallogr D Biol Crystallogr 66:486–501. a nonenveloped virus reveals a priming mechanism for cell entry. Cell 141:472–482. 36. Brünger AT, et al. (1998) Crystallography & NMR System: A new software suite for 19. Liu H, et al. (2010) Atomic structure of human adenovirus by cryo-EM reveals macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54: interactions among protein networks. Science 329:1038–1043. 905–921.

9642 | www.pnas.org/cgi/doi/10.1073/pnas.1018104108 Ge and Zhou Downloaded by guest on September 27, 2021