Joanita Jakana - Academia.edu (original) (raw)

Papers by Joanita Jakana

Research paper thumbnail of 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Proceedings of the National Academy of Sciences, 2010

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin contain... more The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

Research paper thumbnail of Zernike Phase Contrast Cryo-EM for Biological Structure Determination

Microscopy and Microanalysis, 2010

Research paper thumbnail of Zernike Phase Contrast Cryo-Electron Microscopy of Virus Particles

Microscopy and Microanalysis, 2012

Research paper thumbnail of Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

Proceedings of the National Academy of Sciences of the United States of America, Jan 11, 2016

Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, onl... more Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the s...

Research paper thumbnail of Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy 1 1 Edited by W. Baumeister

J Mol Biol, 2000

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The... more Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 Å structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.

Research paper thumbnail of Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300kV

Journal of Structural Biology, 2006

CCD cameras have numerous advantages over photographic Wlm for detecting electrons; however the p... more CCD cameras have numerous advantages over photographic Wlm for detecting electrons; however the point spread function of these cameras has not been suYcient for single particle data collection to subnanometer resolution with 300 kV microscopes. We have adopted spectral signal to noise ratio (SNR) as a parameter for assessing detector quality for single particle imaging. The robustness of this parameter is conWrmed under a variety of experimental conditions. Using this parameter, we demonstrate that the SNR of images of either amorphous carbon Wlm or ice embedded virus particles collected on a new commercially available 4kx4k CCD camera are slightly better than photographic Wlm at low spatial frequency (<1/5 Nyquist frequency), and as good as photographic Wlm out to half of the Nyquist frequency. In addition it is slightly easier to visualize ice embedded particles on this CCD camera than on photographic Wlm. Based on this analysis it is realistic to collect images containing subnanometer resolution data (6-9 Å) using this CCD camera at an eVective magni-Wcation of »112 000£ on a 300 kV electron microscope.

Research paper thumbnail of Seeing the Herpesvirus Capsid at 8.5��

Science, 2000

Human herpesviruses are large and structurally complex viruses that cause a variety of diseases. ... more Human herpesviruses are large and structurally complex viruses that cause a variety of diseases. The three-dimensional structure of the herpesvirus capsid has been determined at 8.5 angstrom resolution by electron cryomicroscopy. More than 30 putative α helices were identified in the four proteins that make up the 0.2 billion-dalton shell. Some of these helices are located at domains that undergo conformational changes during capsid assembly and DNA packaging. The unique spatial arrangement of the heterotrimer at the local threefold positions accounts for the asymmetric interactions with adjacent capsid components and the unusual co-dependent folding of its subunits.

Research paper thumbnail of Three-Dimensional Structure of the Acrosomal Filament of Limulus Sperm by 400 KV Electron Cryomicroscopy. Discussion

Biophysical Journal, May 1, 1995

The acrosomal bundle of Limulus sperm was imaged by electron cryomicroscopy, and the three-dimens... more The acrosomal bundle of Limulus sperm was imaged by electron cryomicroscopy, and the three-dimensional structure of a filament computationally isolated from the bundle was determined by helical image reconstruction. The actin model of Holmes was fit into the map, and its interactions with scruin, the actin-binding and cross-linking protein, were studied. Scruin binds to two consecutive actins along the helix via subdomains 1 and 3. These interactions involve helix-loop-beta motifs that are present in both actin subdomains (in different monomers) in positions available for binding by the same scruin molecule as it wraps around the actin. Taking first the structural motif homology and then looking for sequence pattern similarities, a stretch of 12 out of 20 matches in hydrophobicity is found. Scruin itself has been found to have an internal tandem homology.

Research paper thumbnail of 4.0 � Cryo-EM Structure of the Mammalian Chaperonin: TRiC/CCT

Research paper thumbnail of The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle1

Journal of Molecular Biology, Nov 19, 1999

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 mm process ca... more Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 mm process called the true discharge. The core of this structure is a bundle of ordered ®laments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actinscruin helix that is azimuthally modulated by the in¯uence of the interactions of a ®lament with its neighbors. There are a variety of density connections with neighboring ®laments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three inter®lament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.

Research paper thumbnail of A 7-� Projection Map of Frozen, Hydrated Acrosomal Bundle from Limulus Sperm

Research paper thumbnail of Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum1

Journal of Molecular Biology, Apr 12, 2002

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member o... more VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their¯exible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.

Research paper thumbnail of The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle 1 1 Edited by W. Baumeister

J Mol Biol, 1999

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 μm process ca... more Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 μm process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actin-scruin helix that is azimuthally modulated by the influence of the interactions of a filament with its neighbors. There are a variety of density connections with neighboring filaments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three interfilament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.

Research paper thumbnail of Imaging Frozen, Hydrated Acrosomal Bundle from Limulus Sperm at 7 Å Resolution with a 400 kV Electron Cryomicroscope

Journal of Molecular Biology, Mar 20, 1993

Research paper thumbnail of Identification of the sites of interaction between the scaffold and outer shell in herpes simplex virus-1 capsids by difference electron imaging

Proceedings of the National Academy of Sciences, Mar 17, 1998

Formation of herpes simplex virus-1 capsids requires the presence of intact scaffolding proteins.... more Formation of herpes simplex virus-1 capsids requires the presence of intact scaffolding proteins. The C terminus of the abundant scaffolding protein associates with the major capsid shell protein VP5 through hydrophobic interactions. After cleavage by the viral encoded protease, which removes their C-terminal 25 aa, the scaffolding proteins are released from the capsid. We have used electron cryomicroscopy and computer image processing to determine, to 13 Å, the three-dimensional structures of capsids containing either cleaved or uncleaved scaffolding proteins. Detailed comparisons show that the structures of the outer icosahedral shells are almost identical in the two capsid types. Differences are apparent in the radial distribution of the density inside the capsid shell (within a radius of 460 Å) which represents the scaffolding core. However, in both capsid types, the bulk of this internal density exhibits no icosahedral symmetry. Close examination revealed localized regions of icosahedrally arranged extra density at the interface between the outer shell and the scaffold of protease-minus capsids. Rod-like densities extending inwards for ≈40 Å from the capsid shell are present under four of the six quasi-equivalent triplex positions. Under triplexes Tb, Tc, and Te, the major additional densities appear as pairs with the rods in each pair situated 37 Å apart. We propose that these rods are formed by the C-termini of the scaffolding proteins and represent the sites of interaction between the capsid shell and scaffold.

Research paper thumbnail of Visualization of biological nano-machines at subnanometer resolutions

Research paper thumbnail of Backbone structure of the infectious ε15 virus capsid revealed by electron cryomicroscopy

Nature, 2008

A half-century after the determination of the first threedimensional crystal structure of a prote... more A half-century after the determination of the first threedimensional crystal structure of a protein 1 , more than 40,000 structures ranging from single polypeptides to large assemblies have been reported 2 . The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-Å resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (e15) particle, by singleparticle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity 3,4 . However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond e15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

Research paper thumbnail of Conformational Changes of Eukaryotic Chaperonin TRiC/CCT in the Nucleotide Cycle Revealed by CryoEM

The Faseb Journal, Apr 1, 2009

Research paper thumbnail of Rocking Motion of the Equatorial Domains of a Group II Chaperonin between Two Biochemical States Revealed by Single-Particle Cryo-EM at Near-atomic and Subnanometer Resolutions

The Faseb Journal, Apr 1, 2009

Research paper thumbnail of Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum 1 1 Edited by D. Rees

J Mol Biol, 2002

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member o... more VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.

Research paper thumbnail of 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Proceedings of the National Academy of Sciences, 2010

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin contain... more The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s cellular substrate specificity.

Research paper thumbnail of Zernike Phase Contrast Cryo-EM for Biological Structure Determination

Microscopy and Microanalysis, 2010

Research paper thumbnail of Zernike Phase Contrast Cryo-Electron Microscopy of Virus Particles

Microscopy and Microanalysis, 2012

Research paper thumbnail of Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

Proceedings of the National Academy of Sciences of the United States of America, Jan 11, 2016

Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, onl... more Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the s...

Research paper thumbnail of Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy 1 1 Edited by W. Baumeister

J Mol Biol, 2000

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The... more Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 Å structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.

Research paper thumbnail of Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300kV

Journal of Structural Biology, 2006

CCD cameras have numerous advantages over photographic Wlm for detecting electrons; however the p... more CCD cameras have numerous advantages over photographic Wlm for detecting electrons; however the point spread function of these cameras has not been suYcient for single particle data collection to subnanometer resolution with 300 kV microscopes. We have adopted spectral signal to noise ratio (SNR) as a parameter for assessing detector quality for single particle imaging. The robustness of this parameter is conWrmed under a variety of experimental conditions. Using this parameter, we demonstrate that the SNR of images of either amorphous carbon Wlm or ice embedded virus particles collected on a new commercially available 4kx4k CCD camera are slightly better than photographic Wlm at low spatial frequency (<1/5 Nyquist frequency), and as good as photographic Wlm out to half of the Nyquist frequency. In addition it is slightly easier to visualize ice embedded particles on this CCD camera than on photographic Wlm. Based on this analysis it is realistic to collect images containing subnanometer resolution data (6-9 Å) using this CCD camera at an eVective magni-Wcation of »112 000£ on a 300 kV electron microscope.

Research paper thumbnail of Seeing the Herpesvirus Capsid at 8.5��

Science, 2000

Human herpesviruses are large and structurally complex viruses that cause a variety of diseases. ... more Human herpesviruses are large and structurally complex viruses that cause a variety of diseases. The three-dimensional structure of the herpesvirus capsid has been determined at 8.5 angstrom resolution by electron cryomicroscopy. More than 30 putative α helices were identified in the four proteins that make up the 0.2 billion-dalton shell. Some of these helices are located at domains that undergo conformational changes during capsid assembly and DNA packaging. The unique spatial arrangement of the heterotrimer at the local threefold positions accounts for the asymmetric interactions with adjacent capsid components and the unusual co-dependent folding of its subunits.

Research paper thumbnail of Three-Dimensional Structure of the Acrosomal Filament of Limulus Sperm by 400 KV Electron Cryomicroscopy. Discussion

Biophysical Journal, May 1, 1995

The acrosomal bundle of Limulus sperm was imaged by electron cryomicroscopy, and the three-dimens... more The acrosomal bundle of Limulus sperm was imaged by electron cryomicroscopy, and the three-dimensional structure of a filament computationally isolated from the bundle was determined by helical image reconstruction. The actin model of Holmes was fit into the map, and its interactions with scruin, the actin-binding and cross-linking protein, were studied. Scruin binds to two consecutive actins along the helix via subdomains 1 and 3. These interactions involve helix-loop-beta motifs that are present in both actin subdomains (in different monomers) in positions available for binding by the same scruin molecule as it wraps around the actin. Taking first the structural motif homology and then looking for sequence pattern similarities, a stretch of 12 out of 20 matches in hydrophobicity is found. Scruin itself has been found to have an internal tandem homology.

Research paper thumbnail of 4.0 � Cryo-EM Structure of the Mammalian Chaperonin: TRiC/CCT

Research paper thumbnail of The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle1

Journal of Molecular Biology, Nov 19, 1999

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 mm process ca... more Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 mm process called the true discharge. The core of this structure is a bundle of ordered ®laments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actinscruin helix that is azimuthally modulated by the in¯uence of the interactions of a ®lament with its neighbors. There are a variety of density connections with neighboring ®laments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three inter®lament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.

Research paper thumbnail of A 7-� Projection Map of Frozen, Hydrated Acrosomal Bundle from Limulus Sperm

Research paper thumbnail of Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum1

Journal of Molecular Biology, Apr 12, 2002

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member o... more VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their¯exible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.

Research paper thumbnail of The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle 1 1 Edited by W. Baumeister

J Mol Biol, 1999

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 μm process ca... more Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 μm process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actin-scruin helix that is azimuthally modulated by the influence of the interactions of a filament with its neighbors. There are a variety of density connections with neighboring filaments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three interfilament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.

Research paper thumbnail of Imaging Frozen, Hydrated Acrosomal Bundle from Limulus Sperm at 7 Å Resolution with a 400 kV Electron Cryomicroscope

Journal of Molecular Biology, Mar 20, 1993

Research paper thumbnail of Identification of the sites of interaction between the scaffold and outer shell in herpes simplex virus-1 capsids by difference electron imaging

Proceedings of the National Academy of Sciences, Mar 17, 1998

Formation of herpes simplex virus-1 capsids requires the presence of intact scaffolding proteins.... more Formation of herpes simplex virus-1 capsids requires the presence of intact scaffolding proteins. The C terminus of the abundant scaffolding protein associates with the major capsid shell protein VP5 through hydrophobic interactions. After cleavage by the viral encoded protease, which removes their C-terminal 25 aa, the scaffolding proteins are released from the capsid. We have used electron cryomicroscopy and computer image processing to determine, to 13 Å, the three-dimensional structures of capsids containing either cleaved or uncleaved scaffolding proteins. Detailed comparisons show that the structures of the outer icosahedral shells are almost identical in the two capsid types. Differences are apparent in the radial distribution of the density inside the capsid shell (within a radius of 460 Å) which represents the scaffolding core. However, in both capsid types, the bulk of this internal density exhibits no icosahedral symmetry. Close examination revealed localized regions of icosahedrally arranged extra density at the interface between the outer shell and the scaffold of protease-minus capsids. Rod-like densities extending inwards for ≈40 Å from the capsid shell are present under four of the six quasi-equivalent triplex positions. Under triplexes Tb, Tc, and Te, the major additional densities appear as pairs with the rods in each pair situated 37 Å apart. We propose that these rods are formed by the C-termini of the scaffolding proteins and represent the sites of interaction between the capsid shell and scaffold.

Research paper thumbnail of Visualization of biological nano-machines at subnanometer resolutions

Research paper thumbnail of Backbone structure of the infectious ε15 virus capsid revealed by electron cryomicroscopy

Nature, 2008

A half-century after the determination of the first threedimensional crystal structure of a prote... more A half-century after the determination of the first threedimensional crystal structure of a protein 1 , more than 40,000 structures ranging from single polypeptides to large assemblies have been reported 2 . The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-Å resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (e15) particle, by singleparticle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity 3,4 . However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond e15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

Research paper thumbnail of Conformational Changes of Eukaryotic Chaperonin TRiC/CCT in the Nucleotide Cycle Revealed by CryoEM

The Faseb Journal, Apr 1, 2009

Research paper thumbnail of Rocking Motion of the Equatorial Domains of a Group II Chaperonin between Two Biochemical States Revealed by Single-Particle Cryo-EM at Near-atomic and Subnanometer Resolutions

The Faseb Journal, Apr 1, 2009

Research paper thumbnail of Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum 1 1 Edited by D. Rees

J Mol Biol, 2002

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member o... more VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.