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Cryotomography of Pleomorphic Viruses
574a
Wednesday, February 15, 2017
Fourier-Bessel approach. Progress in EM has been great since then, particularly over the past three to four years with the advent of direct electron detectors. Using the Iterative Helical Real Space Reconstruction method implemented in SPIDER, we have been able to use images of the T4 baseplate assembly to generate reconstructions of the tail tube at better than ˚ resolution, a 1,000-fold improvement in information content over 3.5 A what was possible in 1968. Surprisingly, we can show that a reasonable ˚ 2, using a reconstruction is possible with a dose of only ~ 1.5 electrons/A higher dose set of frames for alignment. Considering that SPIDER was largely written more than 20 years ago, for some specimens at least legacy software may not be a limiting factor. To test this, a comparison will be made with Relion 2.0, a new version of Relion which allows for helical reconstruction. 2822-Pos Board B429 Strain Between Leading and Trailing Heads of a Stepping Kinesin Dimer Visualized in 3D by Cryo-EM Daifei Liu1, Xueqi Liu1, Zhiguo Shang2, Charles V. Sindelar1. 1 Yale University, New Haven, CT, USA, 2UT Southwestern Medical Center, Dallas, TX, USA. The structural basis of walking by dimeric molecules of kinesin along microtubules has remained unclear, partly because available structural methods have been unable to capture microtubule-bound intermediates of this process. We developed a novel method, FINDKIN, that allowed us to solve sub-nanometer resolution cryo-EM maps in which the two heads of a kinesin dimer are attached at sequential sites along a single protofilament of the microtubule, in a stepping configuration. The resulting structures indicate that the upper half of the nucleotide cleft shifts downward in the trailing head and upward in the leading head. Consequently, closure of the nucleotide cleft in the trailing head supports the binding of an ATP analog, while opening of the cleft in the leading head is accompanied by loss of nucleotide density. These results supply a detailed explanation for how tension between the two heads of dimeric kinesin keeps the enzymatic cycles of the heads out of phase in order to stimulate directional motility. Moreover, the novel cryoEM image-processing method presented here paves the way for future structural studies of a variety of challenging systems that bind to microtubules and other biological filaments. 2823-Pos Board B430 Comparison of 3-D Cell and Tissue Imaging Techniques Based on Scanning Electron Probes Emma L. McBride1, Amith Rao1, Guofeng Zhang1, Irina D. Pokrovskaya2, Maria A. Aronova1, Brian Storrie2, Richard D. Leapman1. 1 NIBIB, National Institutes of Health, Bethesda, MD, USA, 2Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA. The use of scanned electron probes, rather than the wide-beam illumination of standard transmission electron microscopy, allows the structural biologist to image 3-D cellular and tissue ultrastructure by taking full advantage of the physical interactions between the incoming electrons and the specimen. For example, in serial block face scanning electron microscopy (SBF-SEM), a low-energy (~1 keV electron probe) produces a backscattered electron signal originating from a thin approximately 25-nm layer below the face of a heavy-atom stained, resin-embedded block. However, due to the low average atomic number of the atoms in the block, the scattering is in the forward direction providing a lateral resolution of around 5 nm. By cutting successive layers from the block face, large (> million cubic micrometers) 3-D volumes of a biological sample can be generated. Bright-field electron tomography in the scanning transmission electron microscope (STEM) is performed with a high-energy (~300 keV) electron probe, which is focused to a 2-nm diameter probe using a low convergence angle, which provides a depth of field of ~2 micrometers in a thick section of a stained, embedded specimen. Unlike in conventional transmission electron microscopy, there are no post-specimen lenses in STEM, so that chromatic aberration does not affect image quality due to multiple inelastic scattering; however, it is necessary to limit the concentration of the heavy-atom stain to avoid attenuation of the probe by elastic scattering. We have applied both SBF-SEM and STEM tomography extensively to determine cellular ultrastructure. In the present study we compare performance and relative advantages of the two techniques in terms of spatial resolution, specimen size, and speed of acquisition. For example, we present data from human blood platelets and secretory cells in pancreatic islets of Langerhans. This work was supported by the Intramural Research Program of the National Institutes of Biomedical Imaging and Bioengineering, NIH.
2824-Pos Board B431 Cryotomography of Pleomorphic Viruses Amar D. Parvate1, Jason Lanman1, Colleen Jonsson2. 1 Biological Sciences, Purdue University, West Lafayette, IN, USA, 2 Department of Microbiology, University of Tennessee, Knoxville, Knoxville, TN, USA. 61st Annual Meeting of Biophysical Society Hantaviruses belong to the Bunyaviridae family and constitute a group of human pathogens causing hemorrhagic fevers with 15-40% mortality. Currently no vaccine is available for these infections, and treatment only relieves symptoms. The Gn-Gc glycoprotein complex on the virus surface is involved in binding to host cell receptors and fusion with host membrane to release the viral genome. It has been postulated that Bunyavirus glycoproteins may belong to class II fusion proteins. Our objective is to determine whether the arrangement of the fusion proteins is similar to that of other class II fusion proteins such as those of Flaviviruses. Using cryo-electron tomography and sub-volume aver˚ resoluaging we propose to solve the structure of Hantavirus spike to 15-20 A tion. Hantaviruses are classified as BSL3 agents. We have optimized a method to purify and inactivate Orthobunyavirus (a BSL2 virus) using 1% glutaraldehyde for cryoET imaging in a BSL1 containment facility. This method was replicated in a BSL3 facility to prepare and inactivate Andes virus (a Hantavirus) samples for cryo-ET. The Andes virus particles were classified as round, elongated and irregular based on tomographic data. Further observations revealed the arrangement of glycoprotein spikes was clearly visible on the surface of both glutaraldehyde fixed viruses, possibly with a 4-fold symmetry in Andes, as in case of Hantaan and Tula virus. 2825-Pos Board B432 Endophilin-Dynamin Complex Assembly - A General Mechanism of Membrane Remodeling Control Anna C. Sundborger1, Veer Bhatt1, Robert Ashley1, Jenny E. Hinshaw2. 1 The Hormel Institute, Austin, MN, USA, 2LCMB, National Institutes of Diabetes & Digestive & Kidney Diseases, Bethesda, MD, USA. Endophilin belongs to a group of proteins containing membrane binding and bending BAR domains. Neuronal-specific endophilin A1 mediates membrane bending in association with dynamin 1-catalyzed membrane fission. In a previous study, we show that endophilin A1 co-localizes with dynamin 1 on necks of clathrin-coated pits in nerve terminals and assembles into a complex with dynamin 1 on tubulated liposomes in vitro. Using cryo-EM and iterative helical real space reconstruction (IHRSR) we have generated a preliminary 3D density map of the endophilin A1–dynamin 1 complex. Our data suggests that endophilin A1 may regulate dynamin 1-catalyzed membrane fission by preventing inter-molecular interactions within the dynamin scaffold. Such interactions promote dynamin stimulated GTPase activity and trigger fission. Thus, endophilin A1 may function as a negative regulator of dynamin 1-catalyzed plasma membrane fission by means of controlling dynamin scaffold organization. This notion is further supported by in vitro observations. Endophilin B1 is a tumor suppressor involved in regulation of mitochondrial dynamics and Bax-mediated apoptosis. Loss of Endophilin B1 was recently shown to inhibit dynamin 2-catalyzed fragmentation of Atg9 vesicles from Golgi during autophagosome formation. This suggests that endophilin B1 may act as a negative regulator of dynamin 2 via formation of an endophilin B1-dynamin 2 complex, in a manner similar to endophilin A1. This further implies a general mechanism for membrane remodeling control, by coordinating membrane bending and fission through endophilin-dynamin complex assembly. In this study, we show that endophilin B1 and dynamin 2 indeed interact in vitro. We aim to solve the structure of the endophilin-dynamin complexes at atomic resolutions using a cryo-EM. Determining the organization of these protein complexes will shed light on potentially general mechanisms that control intracellular membrane remodeling during essential cellular processes, such as apoptosis, endocytosis and autophagy, and identify novel targets for drug discovery. 2826-Pos Board B433 Sub-Surface Serial Block Face SEM of Biological Structures at Near Isotropic Spatial Resolution Qianping He1, David C. Joy2,3, Guofeng Zhang1, Richard D. Leapman1. 1 National Institute of Health, Bethesda, MD, USA, 2Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN, USA, 3 Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN, USA. Serial block face scanning electron microscopy (SBF-SEM) provides nanoscale 3D ultrastructure of entire cells and tissue volumes. In SBF-SEM, an ultramicrotome built into the SEM specimen stage successively removes thin
Wednesday, February 15, 2017
Fourier-Bessel approach. Progress in EM has been great since then, particularly over the past three to four years with the advent of direct electron detectors. Using the Iterative Helical Real Space Reconstruction method implemented in SPIDER, we have been able to use images of the T4 baseplate assembly to generate reconstructions of the tail tube at better than ˚ resolution, a 1,000-fold improvement in information content over 3.5 A what was possible in 1968. Surprisingly, we can show that a reasonable ˚ 2, using a reconstruction is possible with a dose of only ~ 1.5 electrons/A higher dose set of frames for alignment. Considering that SPIDER was largely written more than 20 years ago, for some specimens at least legacy software may not be a limiting factor. To test this, a comparison will be made with Relion 2.0, a new version of Relion which allows for helical reconstruction. 2822-Pos Board B429 Strain Between Leading and Trailing Heads of a Stepping Kinesin Dimer Visualized in 3D by Cryo-EM Daifei Liu1, Xueqi Liu1, Zhiguo Shang2, Charles V. Sindelar1. 1 Yale University, New Haven, CT, USA, 2UT Southwestern Medical Center, Dallas, TX, USA. The structural basis of walking by dimeric molecules of kinesin along microtubules has remained unclear, partly because available structural methods have been unable to capture microtubule-bound intermediates of this process. We developed a novel method, FINDKIN, that allowed us to solve sub-nanometer resolution cryo-EM maps in which the two heads of a kinesin dimer are attached at sequential sites along a single protofilament of the microtubule, in a stepping configuration. The resulting structures indicate that the upper half of the nucleotide cleft shifts downward in the trailing head and upward in the leading head. Consequently, closure of the nucleotide cleft in the trailing head supports the binding of an ATP analog, while opening of the cleft in the leading head is accompanied by loss of nucleotide density. These results supply a detailed explanation for how tension between the two heads of dimeric kinesin keeps the enzymatic cycles of the heads out of phase in order to stimulate directional motility. Moreover, the novel cryoEM image-processing method presented here paves the way for future structural studies of a variety of challenging systems that bind to microtubules and other biological filaments. 2823-Pos Board B430 Comparison of 3-D Cell and Tissue Imaging Techniques Based on Scanning Electron Probes Emma L. McBride1, Amith Rao1, Guofeng Zhang1, Irina D. Pokrovskaya2, Maria A. Aronova1, Brian Storrie2, Richard D. Leapman1. 1 NIBIB, National Institutes of Health, Bethesda, MD, USA, 2Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA. The use of scanned electron probes, rather than the wide-beam illumination of standard transmission electron microscopy, allows the structural biologist to image 3-D cellular and tissue ultrastructure by taking full advantage of the physical interactions between the incoming electrons and the specimen. For example, in serial block face scanning electron microscopy (SBF-SEM), a low-energy (~1 keV electron probe) produces a backscattered electron signal originating from a thin approximately 25-nm layer below the face of a heavy-atom stained, resin-embedded block. However, due to the low average atomic number of the atoms in the block, the scattering is in the forward direction providing a lateral resolution of around 5 nm. By cutting successive layers from the block face, large (> million cubic micrometers) 3-D volumes of a biological sample can be generated. Bright-field electron tomography in the scanning transmission electron microscope (STEM) is performed with a high-energy (~300 keV) electron probe, which is focused to a 2-nm diameter probe using a low convergence angle, which provides a depth of field of ~2 micrometers in a thick section of a stained, embedded specimen. Unlike in conventional transmission electron microscopy, there are no post-specimen lenses in STEM, so that chromatic aberration does not affect image quality due to multiple inelastic scattering; however, it is necessary to limit the concentration of the heavy-atom stain to avoid attenuation of the probe by elastic scattering. We have applied both SBF-SEM and STEM tomography extensively to determine cellular ultrastructure. In the present study we compare performance and relative advantages of the two techniques in terms of spatial resolution, specimen size, and speed of acquisition. For example, we present data from human blood platelets and secretory cells in pancreatic islets of Langerhans. This work was supported by the Intramural Research Program of the National Institutes of Biomedical Imaging and Bioengineering, NIH.
2824-Pos Board B431 Cryotomography of Pleomorphic Viruses Amar D. Parvate1, Jason Lanman1, Colleen Jonsson2. 1 Biological Sciences, Purdue University, West Lafayette, IN, USA, 2 Department of Microbiology, University of Tennessee, Knoxville, Knoxville, TN, USA. 61st Annual Meeting of Biophysical Society Hantaviruses belong to the Bunyaviridae family and constitute a group of human pathogens causing hemorrhagic fevers with 15-40% mortality. Currently no vaccine is available for these infections, and treatment only relieves symptoms. The Gn-Gc glycoprotein complex on the virus surface is involved in binding to host cell receptors and fusion with host membrane to release the viral genome. It has been postulated that Bunyavirus glycoproteins may belong to class II fusion proteins. Our objective is to determine whether the arrangement of the fusion proteins is similar to that of other class II fusion proteins such as those of Flaviviruses. Using cryo-electron tomography and sub-volume aver˚ resoluaging we propose to solve the structure of Hantavirus spike to 15-20 A tion. Hantaviruses are classified as BSL3 agents. We have optimized a method to purify and inactivate Orthobunyavirus (a BSL2 virus) using 1% glutaraldehyde for cryoET imaging in a BSL1 containment facility. This method was replicated in a BSL3 facility to prepare and inactivate Andes virus (a Hantavirus) samples for cryo-ET. The Andes virus particles were classified as round, elongated and irregular based on tomographic data. Further observations revealed the arrangement of glycoprotein spikes was clearly visible on the surface of both glutaraldehyde fixed viruses, possibly with a 4-fold symmetry in Andes, as in case of Hantaan and Tula virus. 2825-Pos Board B432 Endophilin-Dynamin Complex Assembly - A General Mechanism of Membrane Remodeling Control Anna C. Sundborger1, Veer Bhatt1, Robert Ashley1, Jenny E. Hinshaw2. 1 The Hormel Institute, Austin, MN, USA, 2LCMB, National Institutes of Diabetes & Digestive & Kidney Diseases, Bethesda, MD, USA. Endophilin belongs to a group of proteins containing membrane binding and bending BAR domains. Neuronal-specific endophilin A1 mediates membrane bending in association with dynamin 1-catalyzed membrane fission. In a previous study, we show that endophilin A1 co-localizes with dynamin 1 on necks of clathrin-coated pits in nerve terminals and assembles into a complex with dynamin 1 on tubulated liposomes in vitro. Using cryo-EM and iterative helical real space reconstruction (IHRSR) we have generated a preliminary 3D density map of the endophilin A1–dynamin 1 complex. Our data suggests that endophilin A1 may regulate dynamin 1-catalyzed membrane fission by preventing inter-molecular interactions within the dynamin scaffold. Such interactions promote dynamin stimulated GTPase activity and trigger fission. Thus, endophilin A1 may function as a negative regulator of dynamin 1-catalyzed plasma membrane fission by means of controlling dynamin scaffold organization. This notion is further supported by in vitro observations. Endophilin B1 is a tumor suppressor involved in regulation of mitochondrial dynamics and Bax-mediated apoptosis. Loss of Endophilin B1 was recently shown to inhibit dynamin 2-catalyzed fragmentation of Atg9 vesicles from Golgi during autophagosome formation. This suggests that endophilin B1 may act as a negative regulator of dynamin 2 via formation of an endophilin B1-dynamin 2 complex, in a manner similar to endophilin A1. This further implies a general mechanism for membrane remodeling control, by coordinating membrane bending and fission through endophilin-dynamin complex assembly. In this study, we show that endophilin B1 and dynamin 2 indeed interact in vitro. We aim to solve the structure of the endophilin-dynamin complexes at atomic resolutions using a cryo-EM. Determining the organization of these protein complexes will shed light on potentially general mechanisms that control intracellular membrane remodeling during essential cellular processes, such as apoptosis, endocytosis and autophagy, and identify novel targets for drug discovery. 2826-Pos Board B433 Sub-Surface Serial Block Face SEM of Biological Structures at Near Isotropic Spatial Resolution Qianping He1, David C. Joy2,3, Guofeng Zhang1, Richard D. Leapman1. 1 National Institute of Health, Bethesda, MD, USA, 2Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN, USA, 3 Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN, USA. Serial block face scanning electron microscopy (SBF-SEM) provides nanoscale 3D ultrastructure of entire cells and tissue volumes. In SBF-SEM, an ultramicrotome built into the SEM specimen stage successively removes thin