James Z. Chen

Deformable M-Reps for 3D Medical Image Segmentation

M-reps (formerly called DSLs) are a multiscale medial means for modeling and rendering 3D solid geometry. They are particularly well suited to model anatomic objects and in particular to capture prior geometric information effectively in deformable models segmentation approaches. The representation is based on figural models, which define objects at coarse scale by a hierarchy of figures—each figure generally a slab representing a solid region and its boundary simultaneously. This paper focuses on the use of single figure models to segment objects of relatively simple structure.A single figure is a sheet of medial atoms, which is interpolated from the model formed by a net, i.e., a mesh or chain, of medial atoms (hence the name m-reps), each atom modeling a solid region via not only a position and a width but also a local figural frame giving figural directions and an object angle between opposing, corresponding positions on the boundary implied by the m-rep. The special capability of an m-rep is to provide spatial and orientational correspondence between an object in two different states of deformation. This ability is central to effective measurement of both geometric typicality and geometry to image match, the two terms of the objective function optimized in segmentation by deformable models. The other ability of m-reps central to effective segmentation is their ability to support segmentation at multiple levels of scale, with successively finer precision. Objects modeled by single figures are segmented first by a similarity transform augmented by object elongation, then by adjustment of each medial atom, and finally by displacing a dense sampling of the m-rep implied boundary. While these models and approaches also exist in 2D, we focus on 3D objects.The segmentation of the kidney from CT and the hippocampus from MRI serve as the major examples in this paper. The accuracy of segmentation as compared to manual, slice-by-slice segmentation is reported.

Beam-induced motion of vitrified specimen on holey carbon film.

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.

Atomic model of an infectious rotavirus particle.

Non‐enveloped viruses of different types have evolved distinct mechanisms for penetrating a cellular membrane during infection. Rotavirus penetration appears to occur by a process resembling enveloped‐virus fusion: membrane distortion linked to conformational changes in a viral protein. Evidence for such a mechanism comes from crystallographic analyses of fragments of VP4, the rotavirus‐penetration protein, and infectivity analyses of structure‐based VP4 mutants. We describe here the structure of an infectious rotavirus particle determined by electron cryomicroscopy (cryoEM) and single‐particle analysis at about 4.3 Å resolution. The cryoEM image reconstruction permits a nearly complete trace of the VP4 polypeptide chain, including the positions of most side chains. It shows how the two subfragments of VP4 (VP8* and VP5*) retain their association after proteolytic cleavage, reveals multiple structural roles for the β‐barrel domain of VP5*, and specifies interactions of VP4 with other capsid proteins. The virion model allows us to integrate structural and functional information into a coherent mechanism for rotavirus entry.

Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.

Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM

Rotaviruses, major causes of childhood gastroenteritis, are nonenveloped, icosahedral particles with double-strand RNA genomes. By the use of electron cryomicroscopy and single-particle reconstruction, we have visualized a rotavirus particle comprising the inner capsid coated with the trimeric outer-layer protein, VP7, at a resolution (4 Å) comparable with that of X-ray crystallography. We have traced the VP7 polypeptide chain, including parts not seen in its X-ray crystal structure. The 3 well-ordered, 30-residue, N-terminal “arms” of each VP7 trimer grip the underlying trimer of VP6, an inner-capsid protein. Structural differences between free and particle-bound VP7 and between free and VP7-coated inner capsids may regulate mRNA transcription and release. The Ca2+-stabilized VP7 intratrimer contact region, which presents important neutralizing epitopes, is unaltered upon capsid binding.

Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy.

The comparison of a pair of electron microscope images recorded at different specimen tilt angles provides a powerful approach for evaluating the quality of images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair images recorded from a range of specimens with different symmetries and molecular masses and show how the analysis can produce valuable information not easily obtained otherwise. We show that the accuracy of orientation determination of individual single particles depends on molecular mass, as expected theoretically since the information in each particle image increases with molecular mass. The angular uncertainty is less than 1° for particles of high molecular mass (∼ 50 MDa), several degrees for particles in the range 1–5 MDa, and tens of degrees for particles below 1 MDa. Orientational uncertainty may be the major contributor to the effective temperature factor (B-factor) describing contrast loss and therefore the maximum resolution of a structure determination. We also made two unexpected observations. Single particles that are known to be flexible showed a wider spread in orientation accuracy, and the orientations of the largest particles examined changed by several degrees during typical low-dose exposures. Smaller particles presumably also reorient during the exposure; hence, specimen movement is a second major factor that limits resolution. Tilt pairs thus enable assessment of orientation accuracy, map quality, specimen motion, and conformational heterogeneity. A convincing tilt-pair parameter plot, where 60% of the particles show a single cluster around the expected tilt axis and tilt angle, provides confidence in a structure determined using electron cryomicroscopy.

Electron cryomicroscopy structure of N‐ethyl maleimide sensitive factor at 11 Å resolution

N‐ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three‐dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP–ADP mixture at 11 Å resolution by electron cryomicroscopy and single‐particle averaging of NSF·α‐SNAP·SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double‐layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF‐N) emerge from the sides of the D1 domain ring. The density corresponding to α‐SNAP and SNAREs is located on the 6‐fold axis of the structure, near the NSF‐N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.

Architecture and assembly of the archaeal Cdc48*20S proteasome.

Significance From microbes to humans, proteolytic machines called proteasomes cleave proteins that are damaged or unnecessary into peptide fragments. Proteasomes minimally consist of the barrel-like 20S peptidase and an AAA+ ring, which harnesses chemical energy to unfold and translocate proteins into the 20S chamber for degradation. Here, we determine the architecture of a recently discovered proteasome, Cdc48⋅20S, by electron microscopy. A continuous axial channel allows translocation through the double AAA+ rings of Cdc48 into the 20S chamber. A model in which dynamic “wobbling” of the AAA+ unfoldase relative to 20S is necessary for function is ruled out for Cdc48⋅20S by electron-microscopy results showing coaxial alignment of Cdc48 and 20S and by the proteolytic activity of cross-linked complexes. ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48⋅20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.

Distinct quaternary structures of the AAA+ Lon protease control substrate degradation

Significance Lon protease degrades unfolded or damaged proteins as well as numerous cellular regulatory proteins. How these different classes of substrates are recognized is poorly understood. We find that Lon hexamers assemble via a matrix of N-domain interactions to form a dodecamer with altered substrate-degradation properties. Access of protein substrates to the degradation machinery in the dodecamer appears to require passage through equatorial portals. As a consequence, large substrates that are efficiently degraded by hexamers seem to be preferentially excluded from dodecamers. This gating mechanism allows the substrate repertoire of Lon to be adjusted by its assembly state. Lon is an ATPase associated with cellular activities (AAA+) protease that controls cell division in response to stress and also degrades misfolded and damaged proteins. Subunits of Lon are known to assemble into ring-shaped homohexamers that enclose an internal degradation chamber. Here, we demonstrate that hexamers of Escherichia coli Lon also interact to form a dodecamer at physiological protein concentrations. Electron microscopy of this dodecamer reveals a prolate structure with the protease chambers at the distal ends and a matrix of N domains forming an equatorial hexamer–hexamer interface, with portals of ∼45 Å providing access to the enzyme lumen. Compared with hexamers, Lon dodecamers are much less active in degrading large substrates but equally active in degrading small substrates. Our results support a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state.

Segmentation of Single-Figure Objects by Deformable M-reps

This paper describes the basis and behavior of segmentation of single figures in 3D by deformable m-reps models. Results are given for the segmentation of kidneys from CT and of hippocampi from MR images. Special focus is made on multi-scale-level stages of segmentation, on intrinsic correspondences under deformation that are provided by m-reps, and on the match against model-relative templates provided by both theoretical edge strength templates and templates derived from training images.