The Current Picture of the Structure and Assembly of Tobacco Mosaic Virus

Butler, P.J.G.

doi:10.1099/0022-1317-65-2-253

Cited by 206 publications

(125 citation statements)

References 91 publications

(104 reference statements)

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“…The diameter of the TMV particle can be estimated to be just below 18 nm for both virions (the side of the square image is 200 pixel, or 115 nm), and the hole between 4.5 and 5 nm. These values agree with the published structural parameters for TMV (24,25) and are used as benchmarks to justify our choices of half-widths described in the reconstruction protocol above. Although the cylindrical hole is visible in both FBP (left) and the ELT reconstructions, the boundaries are more prominent in ELT.…”

Section: Examplessupporting

confidence: 75%

“…The TMV is made up of a helical cylinder with an outer diameter of ≈18 nm and an inner cylindrical hole along the helical axis with a diameter of ≈4 nm (24,25). The size and regularity of the TMV deems it a suitable sample for a simple comparison-even at the rather low resolution inherent in this particular experiment at ≈7.5 nm resolution.…”

Section: Examplesmentioning

confidence: 99%

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Electron lambda-tomography

Quinto

Skoglund²,

Öktem³

2009

Proc. Natl. Acad. Sci. U.S.A.

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Filtered back-projection and weighted back-projection have long been the methods of choice within the electron microscopy community for reconstructing the structure of macromolecular assemblies from electron tomography data. Here, we describe electron lambda-tomography, a reconstruction method that enjoys the benefits of the above mentioned methods, namely speed and ease of implementation, but also addresses some of their shortcomings. In particular, compared to these standard methods, electron lambdatomography is less sensitive to artifacts that come from structures outside the region that is being reconstructed, and it can sharpen boundaries.local tomography | limited angle tomography | microlocal analysis T his article describes a reconstruction method applicable to electron tomography (ET). The rigorous mathematical description of the method and its application to ET is given in ref.1. Here we concentrate on the functionality of the method in an experimental setting with tests on real ET data. Furthermore, we derive a heuristic explanation for its advantages and guidelines for its usage. Finally, we compare it with the most widely used methods in the field, namely filtered back-projection (FBP) and weighted back-projection (WBP). In this context, it should be mentioned that other reconstruction methods have also been developed and applied to ET. Iterative methods, such as algebraic reconstruction technique (ART) and simultaneous iterative reconstruction technique (SIRT) (2, 3), became practically applicable to ET only after regularization through early stopping. A clever discretization, based on Kaiser-Bessel window functions (blobs), was combined with strongly over-relaxed ART and then applied to ET data in refs. 4-6. Another approach is based on variational regularization where in refs. 7 and 8 relative entropy regularization is applied to ET. For more on these other approaches and their merits, we refer to refs. 9, (section 10.2), 10, and 11.We begin with a very brief introduction to ET, including a discussion of the various data collection geometries and a mathematical formulation of the structure determination problem in ET. This is followed by a brief outline of the FBP and WBP methods. We then move on to our algorithm, electron lambdatomography (ELT), which is based on two-dimensional lambda tomography (12)(13)(14). However, ELT is also valid for a broad range of three-dimensional data acquisition geometries. It is a method that maintains the main benefits of the FBP and WBP methods, namely speed and ease of implementation, while addressing some of the shortcomings. In particular, ELT is generally less sensitive to artifacts that come from structures outside the region of interest (ROI) than these other methods. We conclude by providing examples of reconstructions obtained by ELT from real and simulated ET data.Basic Notation. We now introduce notation used throughout the paper. We let R denote the set of real numbers and R + the set of positive real numbers. The three-dimensional space is denoted by ...

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Section: Examplessupporting

confidence: 75%

Section: Examplesmentioning

confidence: 99%

Electron lambda-tomography

Quinto

Skoglund²,

Öktem³

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The 5′ RNA can be simultaneously uncoated due to the lower lateral intersubunit affinity between CPs in the absence of calcium. On the other hand, in the viral production stage, the CP oligomers add to the 5′ RNA to elongate the virion (30). Because the inner loop of CP takes an ordered conformation upon RNA binding, the assembly of a longer helix is promoted (11).…”

Section: Discussionmentioning

confidence: 99%

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

Zhou

2011

Proc. Natl. Acad. Sci. U.S.A.

114

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Helical assemblies such as filamentous viruses, flagella, and F-actin represent an important category of structures in biology. As the first discovered virus, tobacco mosaic virus (TMV) was at the center of virus research. Previously, the structure of TMV was solved at atomic detail by X-ray fiber diffraction but only for its dormant or high-calcium-concentration state, not its low-calcium-concentration state, which is relevant to viral assembly and disassembly inside host cells. Here we report a helical reconstruction of TMV in its calcium-free, metastable assembling state at 3.3 Å resolution by cryo electron microscopy, revealing both protein side chains and RNA bases. An atomic model was built de novo showing marked differences from the high-calcium, dormant-state structure. Although it could be argued that there might be inaccuracies in the latter structure derived from X-ray fiber diffraction, these differences can be interpreted as conformational changes effected by calcium-driven switches, a common regulatory mechanism in plant viruses. Our comparisons of the structures of the low-and highcalcium states indicate that hydrogen bonds formed by Asp116 and Arg92 in the place of the calcium ion of the dormant (highcalcium) state might trigger allosteric changes in the RNA basebinding pockets of the coat protein. In turn, the coat protein-RNA interactions in our structure favor an adenine-X-guanine (A*G) motif over the G*A motif of the dormant state, thus offering an explanation underlying viral assembly initiation by an AAG motif.H elical assemblies represent a very important class of biological structures, performing various tasks for the proper functions of life or executing malicious functions in disease. For example, protein helical assemblies such as cytoskeletal networks and muscle fibers are essential components in our bodies (1, 2). Other helical assemblies include bacterial flagella and secretion systems. Helical viruses, such as tobacco mosaic virus (TMV), account for a major fraction of the virus kingdom. Such helical structures, bearing a special kind of 2D periodicity and potential flexibility, are difficult for structural determination to atomic resolution by conventional methods including X-ray crystallography and NMR. Thus, the molecular mechanisms of action in many of these systems are not understood to atomic detail. Even for TMV, the first virus ever isolated and extensively studied (3) by both X-ray diffraction (e.g., 4-8) and cryo electron microscopy (cryoEM) (9, 10), we still do not know its structure at atomic detail in a state that is relevant to its assembly and disassembly processes, and the underlying mechanisms of these processes remain unknown.The viral RNA of TMV is infectious by itself, but RNA on its 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 recognizes the initiation moti...

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“…Depending on the conditions (such as concentration, pH, temperature, ionic strength and composition of the medium) this protein can in solution form a large number of different specific ordered aggregates (for reviews see Butler [1] and Bloomer and Butler [2]). In particular, at pH near 8.0 and low ionic strength TMV CP exists in the form of an equilibrium dynamic mixture of monomers, two-layer trimers and pentamers, which is called A-protein, and at pHi6.0 and moderate ionic strength the protein forms virus-like helical aggregates called repolymerized protein (RP).…”

Section: Introductionmentioning

confidence: 99%

A comparative differential scanning calorimetric study of tobacco mosaic virus and of its coat protein ts mutant

et al. 1998

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The differential scanning calorimetry (DSC) 'melting curves' for virions and coat proteins (CP) of wild-type tobacco mosaic virus (strain UI) and for its CP ts mutant ts21-66 were measured. Strain UI and ts21-66 mutant (two amino acid substitutions in CP: I21 ~ T and D66 ~ G) differ in the type of symptoms they induce on some host plants. It was observed that CP subunits of both U1 and ts21-66 at pH 8.0, in the form of small (3-4S) aggregates, possess much lower thermal stability than in the virions. Assembly into the virus particles resulted in a DSC melting temperature increase from 41 to 72°C for UI and from 38 to 72°C for ts21-66 CP. In the RNA-free helical viruslike protein assemblies UI and ts21-66 CP subunits had a thermal stability intermediate between those in 3--4S aggregates and in the virions, ts21-66 helical protein displayed a somewhat lower thermal stability than U1.

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The Current Picture of the Structure and Assembly of Tobacco Mosaic Virus

Cited by 206 publications

References 91 publications

Electron lambda-tomography

Electron lambda-tomography

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

A comparative differential scanning calorimetric study of tobacco mosaic virus and of its coat protein ts mutant

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