Cryo-atomic Force Microscopy Research Articles

The human IgM pentamer is a mushroom-shaped molecule with a flexural bias.

The textbook planar model of pentameric IgM, a potent activator of complement C1q, is based upon the crystallographic structure of IgG. Although widely accepted, key predictions of this model have not yet been directly confirmed, which is particularly important since IgG lacks a major Ig fold domain in its Fc region that is present in IgM. Here, we construct a homology-based structural model of the IgM pentamer using the recently obtained crystallographic structure of IgE Fc, which has this additional Ig domain, under the constraint that all of the cysteine residues known to form disulfide bridges both within each monomer and between monomers are bonded together. In contrast to the planar model, this model predicts a non-planar, mushroom-shaped complex, with the central portion formed by the C-terminal domains protruding out of the plane formed by the Fab domains. This unexpected conformation of IgM is, however, directly confirmed by cryo-atomic force microscopy of individual human IgM molecules. Further analysis of this model with free energy calculations of out-of-plane Fab domain rotations reveals a pronounced asymmetry favoring flexions toward the central protrusion. This bias, together with polyvalent attachment to cell surface antigen, would ensure that the IgM pentamer is oriented on the cell membrane with its C1q binding sites fully exposed to the solution, and thus provides a mechanistic explanation for the first steps of C1q activation by IgM.

Application of Calorimetry, Sub-Ambient Atomic Force Microscopy and Dynamic Mechanical Analysis to the Study of Frozen Aqueous Trehalose Solutions

Disaccharides such as trehalose are widely used as cryo-protectants to maintain the activity of proteinaceous drugs during freezing. One unresolved issue is the double transition that is observed very commonly in DSC experiments on disaccharide solutions in the frozen state; the assignment of these transitions remains disputed. Here we use calorimetry and two new techniques to shed light on the true nature of these transitions. Modulated Temperature DSC (MTDSC), cryo atomic force microscopy (AFM) and a novel DMA technique were used to study these transitions. MTDSC identified the two transitions Tr1 and Tr2 at -35.4 and -27.9 degrees C respectively in the reversing heat flow signal, an exotherm and endotherm were observed in the non-reversing signal at circa -32 and -29 degrees C respectively. It is shown for the first time that AFM images can be obtained of a softening and melting sample without damaging it. A force modulation imaging technique showed a softening at Tr1 and a loss of ice crystals at Tr2. These observations were supported by the DMA results. The results indicate Tr1 is associated with a glass transition while Tr2 is associated with the onset of loss of crystallinity.

Localization of Linker Histone in Chromatosomes by Cryo-Atomic Force Microscopy

Linker histones play a fundamental role in determining higher order chromatin structure as a consequence of their association with nucelosomal DNA. Yet the locations and structural consequences of linker histone binding are still enigmatic. Here, using cryo-atomic force microscopy, we show that the linker histone H5 in native chromatin and in chromatosomes reconstituted on the 5S rDNA template is located at the dyad of the nucleosome core particle, within the “stem” structure. Direct measurement also indicates that the length of free linker DNA between chromatosomes in native chromatin is ∼30 bp, slightly shorter than that estimated from nuclease digestion assays.

Cryo-atomic Force Microscopy of Unphosphorylated and Thiophosphorylated Single Smooth Muscle Myosin Molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.

Computer Modeling of Three-dimensional Structure of DNA-packaging RNA (pRNA) Monomer, Dimer, and Hexamer of Phi29 DNA Packaging Motor

A striking common feature in the maturation of all linear double-stranded DNA viruses is that their lengthy genome is translocated with remarkable velocity into the limited space within a preformed protein shell and packaged into near crystalline density. A DNA-translocating motor, powered by ATP hydrolysis, accomplishes this task, which would otherwise be energetically unfavorable. DNA-packaging RNA, pRNA, forms a hexameric complex to serve as a vital component of the DNA translocating motor of bacterial virus Phi29. The sequential action of six pRNA ensures continual function in the DNA translocation process. The Phi29 motor has been assembled with purified components synthesized by chemical or biotechnological approaches and is able to pump the viral DNA into the protein shell in vitro. pRNA dimers are the building blocks of the hexamer. The computer models of the three-dimensional structure of the motor was constructed based on experimental data derived from photoaffinity cross-linking by psoralen, phenphi (cis-Rh(1,10-phenanthroline)(9,10-phenan-threnequinone diimine)Cl(2)(+)), and azidophenacyl; chemical modification and chemical modification interference with dimethyl sulfate, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate, and kethoxal; complementary modification; and nuclease probing by single- and double-stranded specific RNases. The shapes of these computer models are very similar to the published pRNA images of cryo-atomic force microscopy. pRNA hexamer docking with the connector crystal structure reveals a very impressive match with the available biochemical, genetic, and physical data.

Three-dimensional Interaction of Phi29 pRNA Dimer Probed by Chemical Modification Interference, Cryo-AFM, and Cross-linking

Six pRNAs (p for packaging) of bacterial virus phi29 form a hexamer complex that is an essential component of the viral DNA translocating motor. Dimers, the building block of pRNA hexamer, assemble in the order of dimer --> tetramer --> hexamer. The two-dimensional structure of the pRNA monomer has been investigated extensively; however, the three-dimensional structure concerning the distance constraints of the three stems and loops are unknown. In this report, we probed the three-dimensional structure of pRNA monomer and dimer by photo affinity cross-linking with azidophenacyl. Bases 75-81 of the left stem were found to be oriented toward the head loop and proximate to bases 26-31 in a parallel orientation. Chemical modification interference indicates the involvement of bases 45-71 and 82-91 in dimer formation. Dimer was formed via hand-in-hand contact, a novel RNA dimerization that in some aspects is similar to the kissing loops of the human immunodeficiency virus. The covalently linked dimers were found to be biologically active. Both the native dimer and the covalently linked dimer were found by cryo-atomic force microscopy to be similar in global conformation and size.

Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification.

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.

A Dimer as a Building Block in Assembling RNA: A HEXAMER THAT GEARS BACTERIAL VIRUS phi29 DNA-TRANSLOCATING MACHINERY

Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryo-atomic force microscopy revealed that monomers displayed a check mark outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg(2+), Ca(2+), and Mn(2+). Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer --> tetramer --> hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.

Cryoatomic Force Microscopy of Filamentous Actin

Cryoatomic force microscopy (cryo-AFM) was used to image phalloidin-stabilized actin filaments adsorbed to mica. The single filaments are clearly shown to be right-handed helical structures with a periodicity of ∼38 nm. Even at a moderate concentration (∼10 μg/ml), narrow, branched rafts of actin filaments and larger aggregates have been observed. The resolution achieved is sufficient to resolve actin monomers within the filaments. A closer examination of the images shows that the branched rafts are composed of up to three individual filaments with a highly regular lateral registration with a fixed axial shift of ∼13 nm. The implications of these higher-order structures are discussed in terms of x-ray fiber diffraction and rheology of actin gels. The cryo-AFM images also indicate that the recently proposed model of left-handed F-actin is likely to be an artifact of preparation and/or low-resolution AFM imaging.

Resolving Spatial Conformations of Immuno-Proteins with Cryo-Atomic Force Microscopy (Cryo-Afm)

Abstract A major advantage of atomic force microscopy (AFM) is its intrinsic high contrast, owing to its extremely high sensitivity to height variations. For clean, flat specimens, high-resolution images of a variety of bio-molecules have been obtained, demonstrating the usefulness of AFM in structural biology. However, for large molecular complexes, the effectiveness of AFM is seriously limited, partly owing to the excessive deformation induced by the probe force exerted on the specimen. To overcome this difficulty, we have developed a cryo-AFM, operated under liquid nitrogen vapor at a temperature only a few degrees above 77K. In this system, specimen or tip contamination is significantly reduced, and the resolution achieved with isolated macromolecules is generally higher than that obtainable at room temperature with AFM. Therefore, cryo-AFM has been proven to be a worthy alternative for conventional electron microscopy (EM) with the potential of higher resolution without image averaging. A particularly fruitful application of cryo-AFM is the study of the spatial structure of several immuno-proteins, where the 3D surface topology of the molecule has been obtained.

Cryo-atomic force microscopy of smooth muscle myosin

The motor and regulatory domains of the head and the 14-nm pitch of the alpha-helical coiled-coil of the tail of extended (6S) smooth-muscle myosin molecules were imaged with cryo atomic force microscopy at 80-85 K, and the effects of thiophosphorylation of the regulatory light chain were examined. The tail was 4 nm shorter in thiophosphorylated than in nonphosphorylated myosin. The first major bend was invariant, at approximately 51 nm from the head-tail junction (H-T), coincident with low probability in the paircoil score. The second major bend was 100 nm from the H-T junction in nonphosphorylated and closer to a skip residue than the bend (at 95 nm) in thiophosphorylated molecules. The shorter tail and distance between the two major bends induced by thiophosphorylation are interpreted to result from melting of the coiled-coil. An additional bend not previously reported occurred, with a lower frequency, approximately 24 nm from the H-T. The range of separation between the two heads was greater in thiophosphorylated molecules. Occasional high-resolution images showed slight unwinding of the coiled-coil of the base of the heads. We suggest that phosphorylation of MLC20 can affect the structure of extended, 6S myosin.

Cryo Atomic Force Microscopy: A New Approach for Biological Imaging at High Resolution

A low-temperature atomic force microscope (cryo-AFM), operated in liquid nitrogen vapor, has been constructed for biological applications. The system provides an adjustable imaging temperature from 77 to 220 K with atomic resolution achieved on crystalline specimens. Imaging with NaCl microcrystals demonstrates that the system is free from surface contamination. Below 100 K, several biological specimens, including immunoglobulins and DNA as well as red blood cell ghosts, were imaged at high spatial resolution. Measurements on individual macromolecules showed that the mechanical strength is significantly greater at cryogenic temperatures with an estimated Young's modulus 1000-10,000 times that of a hydrated protein at room temperature, providing a solid basis for future improvements and applications of cryo-AFM in structural biology.