Leandro F Estrozi - Profile on Academia.edu (original) (raw)
Papers by Leandro F Estrozi
Journal of Structural Biology, 2008
A new FFT-accelerated projection matching method is presented and tested. The electron microscopy... more A new FFT-accelerated projection matching method is presented and tested. The electron microscopy images are represented by their Fourier-Bessel transforms and the 3D model by its expansion in spherical harmonics, or more specifically in terms of symmetry-adapted functions. The rotational and translational properties of these representations are used to quickly access all the possible 2D projections of the 3D model, which allow an exhaustive inspection of the whole five-dimensional domain of parameters associated to each particle.
Journal of Structural Biology, 2010
A protocol to attain high-resolution single-particle reconstructions is presented. The protocol i... more A protocol to attain high-resolution single-particle reconstructions is presented. The protocol is the concatenation of two procedures: one to obtain an ab initio low-resolution reconstruction, the other to determine a fixed point of the consecutive applications of fast projection matching and 3D reconstruction. It is a reciprocal space formulation where the Fourier coefficients of the 3D scattering density are expressed in terms of symmetry adapted functions and the 2D particle images are represented by their Fourier-Bessel transforms. The new protocol shows advantages in terms of speed and accuracy when compared to other methods currently in use. We illustrate its performance as applied to high-resolution cryo-electron micrographs of rotavirus.
Multiresolution shape representation without border shifting
Electronics Letters, 1999
ABSTRACT The authors show how exact dilations can be used to obtain multiresolution skeletons and... more ABSTRACT The authors show how exact dilations can be used to obtain multiresolution skeletons and how a new multiresolution approach to shape representation can be achieved in terms of such skeletons. In addition to allowing the accurate reconstruction of multiresolution versions of the original binary shapes, the proposed approach is characterised by great simplicity and the important property that the shape borders are not shifted as the smoothing increases
Journal of Virology, 2008
Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid ... more Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T ؍ 13 to T ؍ 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.
Digital Signal Processing, 2003
A careful comparison of three numeric techniques for estimation of the curvature along spatially ... more A careful comparison of three numeric techniques for estimation of the curvature along spatially quantized contours is reported. Two of the considered techniques are based on the Fourier transform (operating over 1D and 2D signals) and Gaussian regularization required to attenuate the spatial quantization noise. While the 1D approach has been reported before and used in a series of applications, the 2D Fourier transform-based method is reported in this article for the first time. The third approach, based on splines, represents a more traditional alternative. Three classes of parametric curves are investigated: analytical, B-splines, and synthesized in the Fourier domain. Four quantization schemes are considered: grid intersect quantization, square box quantization, a table scanner, and a video camera. The performances of the methods are evaluated in terms of their execution speed, curvature error, and sensitivity to the involved parameters. The third approach resulted the fastest, but implied larger errors; the Fourier methods allowed higher accuracy and were robust to parameter configurations. The 2D Fourier method provides the curvature values along the whole image, but exhibits interference in some situations. Such results are important not only for characterizing the relative performance of the considered methods, but also for providing practical guidelines for those interested in applying those techniques to real problems.
Structure determination of feline calicivirus virus-like particles in the context of a pseudo-octahedral arrangement
PloS one, 2015
The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahed... more The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P213. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-te...
Structural Similarity of Secretins from Type II and Type III Secretion Systems
Structure, 2014
Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, ... more Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.
Lecture Notes in Computer Science, 2000
A powerful framework for the representation, characterization and analysis of two-dimensional sha... more A powerful framework for the representation, characterization and analysis of two-dimensional shapes, with special attention given to neurons, is presented. This framework is based on a recently reported approach to scale space skeletonization and respective reconstructions by using label propagation and the exact distance transform. This methodology allows a series of remarkable properties, including the obtention of high quality skeletons, scale space representation of the shapes under analysis without border shifting, selection of suitable spatial scales, and the logical hierarchical decomposition of the shapes in terms of basic components. The proposed approach is illustrated with respect to neuromorphometry, including a novel and fully automated approach to automated dendrogram extraction and the characterization of the main properties of the dendritic arborization which, if necessary, can be done in terms of the branching hierarchy. The reported results fully corroborate the simplicity and potential of the proposed concepts and framework for shape characterization and analysis.
Virology, 2008
The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American h... more The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American human trypanosomiasis (Chagas disease) is infected by the Triatoma virus (TrV). TrV has been classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work presents the three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of the TrV capsid at about 25 Å resolution and its use as a template for phasing the available crystallographic data by the molecular replacement method. The main structural differences between the cryo-EM reconstruction of TrV and other two viruses, one from the same family, the cricket paralysis virus (CrPV) and the human rhinovirus 16 from the Picornaviridae family are presented and discussed.
Nature Structural & Molecular Biology, 2011
Journal of Virology, 2008
There are more than 100 known adenovirus (AdV) serotypes, including 50 human serotypes. Because A... more There are more than 100 known adenovirus (AdV) serotypes, including 50 human serotypes. Because AdV-induced disease is relatively species specific, vectors derived from nonhuman serotypes may have wider clinical potential based, in part, on the lack of ubiquitous memory immunity. Whereas a few of the human serotype capsids have been studied at the structural level, none of the nonhuman serotypes has been analyzed. The basis laid by the analysis of human AdV (hAdV) has allowed us to determine and compare the threedimensional structure of the capsid of canine serotype 2 (CAV-2) to that of hAdV serotype 5 (hAdV-5). We show that CAV-2 capsid has a smoother structure than the human serotypes. Many of the external loops found in the hAdV-5 penton base and the hexon, against which the antibody response is directed, are shorter or absent in CAV-2. On the other hand, the CAV-2 fiber appears to be more complex, with two bends in the shaft. An interesting difference between the human and canine viruses is that the C-terminal part of protein IX is in a different position, making an antenna sticking out of the CAV-2 capsid. The comparison between the two viruses allows the identification of sites that should be easy to modify on the CAV-2 capsid for altering tissue tropism or other biological activities.
Journal of Structural Biology, 2007
A method for finding the center of cryo-EM images which correspond to the projections of a symmet... more A method for finding the center of cryo-EM images which correspond to the projections of a symmetric 3D structure, based on mathematical properties of symmetry adapted functions and the Fourier-Bessel transform, is presented. It is a model independent one-step procedure with no parameters to be chosen by the user. The proposed method is tested in one synthetic tetrahedral case with different noise levels and in two real cases with D 7 and icosahedral symmetries.
The cryo-EM Reconstruction of Drosophila C Virus (DCV) at 5.4 Å
Biophysical Journal, 2013
Biochimie, 2012
The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covere... more The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.
Journal of Structural Biology, 2008
A new FFT-accelerated projection matching method is presented and tested. The electron microscopy... more A new FFT-accelerated projection matching method is presented and tested. The electron microscopy images are represented by their Fourier-Bessel transforms and the 3D model by its expansion in spherical harmonics, or more specifically in terms of symmetry-adapted functions. The rotational and translational properties of these representations are used to quickly access all the possible 2D projections of the 3D model, which allow an exhaustive inspection of the whole five-dimensional domain of parameters associated to each particle.
Journal of Structural Biology, 2010
A protocol to attain high-resolution single-particle reconstructions is presented. The protocol i... more A protocol to attain high-resolution single-particle reconstructions is presented. The protocol is the concatenation of two procedures: one to obtain an ab initio low-resolution reconstruction, the other to determine a fixed point of the consecutive applications of fast projection matching and 3D reconstruction. It is a reciprocal space formulation where the Fourier coefficients of the 3D scattering density are expressed in terms of symmetry adapted functions and the 2D particle images are represented by their Fourier-Bessel transforms. The new protocol shows advantages in terms of speed and accuracy when compared to other methods currently in use. We illustrate its performance as applied to high-resolution cryo-electron micrographs of rotavirus.
Multiresolution shape representation without border shifting
Electronics Letters, 1999
ABSTRACT The authors show how exact dilations can be used to obtain multiresolution skeletons and... more ABSTRACT The authors show how exact dilations can be used to obtain multiresolution skeletons and how a new multiresolution approach to shape representation can be achieved in terms of such skeletons. In addition to allowing the accurate reconstruction of multiresolution versions of the original binary shapes, the proposed approach is characterised by great simplicity and the important property that the shape borders are not shifted as the smoothing increases
Journal of Virology, 2008
Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid ... more Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T ؍ 13 to T ؍ 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.
Digital Signal Processing, 2003
A careful comparison of three numeric techniques for estimation of the curvature along spatially ... more A careful comparison of three numeric techniques for estimation of the curvature along spatially quantized contours is reported. Two of the considered techniques are based on the Fourier transform (operating over 1D and 2D signals) and Gaussian regularization required to attenuate the spatial quantization noise. While the 1D approach has been reported before and used in a series of applications, the 2D Fourier transform-based method is reported in this article for the first time. The third approach, based on splines, represents a more traditional alternative. Three classes of parametric curves are investigated: analytical, B-splines, and synthesized in the Fourier domain. Four quantization schemes are considered: grid intersect quantization, square box quantization, a table scanner, and a video camera. The performances of the methods are evaluated in terms of their execution speed, curvature error, and sensitivity to the involved parameters. The third approach resulted the fastest, but implied larger errors; the Fourier methods allowed higher accuracy and were robust to parameter configurations. The 2D Fourier method provides the curvature values along the whole image, but exhibits interference in some situations. Such results are important not only for characterizing the relative performance of the considered methods, but also for providing practical guidelines for those interested in applying those techniques to real problems.
Structure determination of feline calicivirus virus-like particles in the context of a pseudo-octahedral arrangement
PloS one, 2015
The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahed... more The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P213. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-te...
Structural Similarity of Secretins from Type II and Type III Secretion Systems
Structure, 2014
Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, ... more Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.
Lecture Notes in Computer Science, 2000
A powerful framework for the representation, characterization and analysis of two-dimensional sha... more A powerful framework for the representation, characterization and analysis of two-dimensional shapes, with special attention given to neurons, is presented. This framework is based on a recently reported approach to scale space skeletonization and respective reconstructions by using label propagation and the exact distance transform. This methodology allows a series of remarkable properties, including the obtention of high quality skeletons, scale space representation of the shapes under analysis without border shifting, selection of suitable spatial scales, and the logical hierarchical decomposition of the shapes in terms of basic components. The proposed approach is illustrated with respect to neuromorphometry, including a novel and fully automated approach to automated dendrogram extraction and the characterization of the main properties of the dendritic arborization which, if necessary, can be done in terms of the branching hierarchy. The reported results fully corroborate the simplicity and potential of the proposed concepts and framework for shape characterization and analysis.
Virology, 2008
The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American h... more The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American human trypanosomiasis (Chagas disease) is infected by the Triatoma virus (TrV). TrV has been classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work presents the three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of the TrV capsid at about 25 Å resolution and its use as a template for phasing the available crystallographic data by the molecular replacement method. The main structural differences between the cryo-EM reconstruction of TrV and other two viruses, one from the same family, the cricket paralysis virus (CrPV) and the human rhinovirus 16 from the Picornaviridae family are presented and discussed.
Nature Structural & Molecular Biology, 2011
Journal of Virology, 2008
There are more than 100 known adenovirus (AdV) serotypes, including 50 human serotypes. Because A... more There are more than 100 known adenovirus (AdV) serotypes, including 50 human serotypes. Because AdV-induced disease is relatively species specific, vectors derived from nonhuman serotypes may have wider clinical potential based, in part, on the lack of ubiquitous memory immunity. Whereas a few of the human serotype capsids have been studied at the structural level, none of the nonhuman serotypes has been analyzed. The basis laid by the analysis of human AdV (hAdV) has allowed us to determine and compare the threedimensional structure of the capsid of canine serotype 2 (CAV-2) to that of hAdV serotype 5 (hAdV-5). We show that CAV-2 capsid has a smoother structure than the human serotypes. Many of the external loops found in the hAdV-5 penton base and the hexon, against which the antibody response is directed, are shorter or absent in CAV-2. On the other hand, the CAV-2 fiber appears to be more complex, with two bends in the shaft. An interesting difference between the human and canine viruses is that the C-terminal part of protein IX is in a different position, making an antenna sticking out of the CAV-2 capsid. The comparison between the two viruses allows the identification of sites that should be easy to modify on the CAV-2 capsid for altering tissue tropism or other biological activities.
Journal of Structural Biology, 2007
A method for finding the center of cryo-EM images which correspond to the projections of a symmet... more A method for finding the center of cryo-EM images which correspond to the projections of a symmetric 3D structure, based on mathematical properties of symmetry adapted functions and the Fourier-Bessel transform, is presented. It is a model independent one-step procedure with no parameters to be chosen by the user. The proposed method is tested in one synthetic tetrahedral case with different noise levels and in two real cases with D 7 and icosahedral symmetries.
The cryo-EM Reconstruction of Drosophila C Virus (DCV) at 5.4 Å
Biophysical Journal, 2013
Biochimie, 2012
The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covere... more The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.