40 results on '"Amedee des Georges"'
Search Results
2. Human parainfluenza virus fusion complex glycoproteins imaged in action on authentic viral surfaces.
- Author
-
Tara C Marcink, Tong Wang, Amedee des Georges, Matteo Porotto, and Anne Moscona
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Infection by human parainfluenza viruses (HPIVs) causes widespread lower respiratory diseases, including croup, bronchiolitis, and pneumonia, and there are no vaccines or effective treatments for these viruses. HPIV3 is a member of the Respirovirus species of the Paramyxoviridae family. These viruses are pleomorphic, enveloped viruses with genomes composed of single-stranded negative-sense RNA. During viral entry, the first step of infection, the viral fusion complex, comprised of the receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) and the fusion glycoprotein (F), mediates fusion upon receptor binding. The HPIV3 transmembrane protein HN, like the receptor-binding proteins of other related viruses that enter host cells using membrane fusion, binds to a receptor molecule on the host cell plasma membrane, which triggers the F glycoprotein to undergo major conformational rearrangements, promoting viral entry. Subsequent fusion of the viral and host membranes allows delivery of the viral genetic material into the host cell. The intermediate states in viral entry are transient and thermodynamically unstable, making it impossible to understand these transitions using standard methods, yet understanding these transition states is important for expanding our knowledge of the viral entry process. In this study, we use cryo-electron tomography (cryo-ET) to dissect the stepwise process by which the receptor-binding protein triggers F-mediated fusion, when forming a complex with receptor-bearing membranes. Using an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids and a series of biological tools that permit the capture of intermediate states in the fusion process, we visualize the series of events that occur when a pristine, authentic viral particle interacts with target receptors and proceeds from the viral entry steps of receptor engagement to membrane fusion.
- Published
- 2020
- Full Text
- View/download PDF
3. Cryo-EM Structure of Mechanosensitive Channel YnaI Using SMA2000: Challenges and Opportunities
- Author
-
Claudio Catalano, Danya Ben-Hail, Weihua Qiu, Paul Blount, Amedee des Georges, and Youzhong Guo
- Subjects
YnaI ,SMA2000 ,NCMN ,cryo-EM ,Mechanosensitive Channel ,Chemical technology ,TP1-1185 ,Chemical engineering ,TP155-156 - Abstract
Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, the complete structure of the transmembrane domain of YnaI was not resolved. This reveals a significant limitation of SMA2000 or similar membrane-active copolymers. This limitation may come from the heterogeneity of the polymers and nonspecific interactions between the polymers and the relatively large hydrophobic pockets within the transmembrane domain of YnaI. However, this limitation offers development opportunities for detergent-free technology for challenging membrane proteins.
- Published
- 2021
- Full Text
- View/download PDF
4. Mechanisms of opening and closing of the bacterial replicative helicase
- Author
-
Jillian Chase, Andrew Catalano, Alex J Noble, Edward T Eng, Paul DB Olinares, Kelly Molloy, Danaya Pakotiprapha, Martin Samuels, Brian Chait, Amedee des Georges, and David Jeruzalmi
- Subjects
DNA replication ,DnaB replicative helicase ,helicase loader ,replication initiation ,cryogenic electron microscopy ,structural biology ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the Escherichia coli DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.
- Published
- 2018
- Full Text
- View/download PDF
5. Viral Entry Properties Required for Fitness in Humans Are Lost through Rapid Genomic Change during Viral Isolation
- Author
-
Sho Iketani, Ryan C. Shean, Marion Ferren, Negar Makhsous, Dolly B. Aquino, Amedee des Georges, Bert Rima, Cyrille Mathieu, Matteo Porotto, Anne Moscona, and Alexander L. Greninger
- Subjects
entry mechanisms ,lung infection ,metagenomics ,parainfluenza virus ,paramyxovirus ,viral evolution ,Microbiology ,QR1-502 - Abstract
IMPORTANCE Human parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions.
- Published
- 2018
- Full Text
- View/download PDF
6. Routine single particle CryoEM sample and grid characterization by tomography
- Author
-
Alex J Noble, Venkata P Dandey, Hui Wei, Julia Brasch, Jillian Chase, Priyamvada Acharya, Yong Zi Tan, Zhening Zhang, Laura Y Kim, Giovanna Scapin, Micah Rapp, Edward T Eng, William J Rice, Anchi Cheng, Carl J Negro, Lawrence Shapiro, Peter D Kwong, David Jeruzalmi, Amedee des Georges, Clinton S Potter, and Bridget Carragher
- Subjects
cryoET ,single particle ,protomo ,fiducial-less ,air-water ,tomography ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.
- Published
- 2018
- Full Text
- View/download PDF
7. Subnanometer structure of an enveloped virus fusion complex on viral surface reveals new entry mechanisms
- Author
-
Tara C. Marcink, Gillian Zipursky, Wenjing Cheng, Kyle Stearns, Shari Stenglein, Kate Golub, Frances Cohen, Francesca Bovier, Daniel Pfalmer, Alexander L. Greninger, Matteo Porotto, Amedee des Georges, and Anne Moscona
- Subjects
Multidisciplinary - Abstract
Paramyxoviruses—including important pathogens like parainfluenza, measles, and Nipah viruses—use a receptor binding protein [hemagglutinin-neuraminidase (HN) for parainfluenza] and a fusion protein (F), acting in a complex, to enter cells. We use cryo–electron tomography to visualize the fusion complex of human parainfluenza virus 3 (HN/F) on the surface of authentic clinical viruses at a subnanometer resolution sufficient to answer mechanistic questions. An HN loop inserts in a pocket on F, showing how the fusion complex remains in a ready but quiescent state until activation. The globular HN heads are rotated with respect to each other: one downward to contact F, and the other upward to grapple cellular receptors, demonstrating how HN/F performs distinct steps before F activation. This depiction of viral fusion illuminates potentially druggable targets for paramyxoviruses and sheds light on fusion processes that underpin wide-ranging biological processes but have not been visualized in situ or at the present resolution.
- Published
- 2023
8. Investigating gating mechanisms of ion channels using temperature-resolved cryoEM
- Author
-
Amedee des Georges, Zephan Melville, Youzhong Guo, Harsh Bansia, Claudio Catalano, and Andrew R. Marks
- Subjects
Chemistry ,Biophysics ,Gating ,Instrumentation ,Ion channel - Published
- 2021
9. Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly
- Author
-
Braden M. Roth, Paul T. Wilder, Christopher Peralta, Richard R. Rustandi, Jessica W. Olson, Xingjian Xu, Sarah L. J. Michel, Wenbo Yu, Mary E. Cook, Alexander D. MacKerell, Heather M. Neu, Kristen M. Varney, Thomas E. Cleveland, Alexander Grishaev, John W. Loughney, Catherine Lancaster, Danya Ben-Hail, E. Pozharski, Adam Kristopeit, Sianny Christanti, Amedee des Georges, Kaylin A. Adipietro, Dorothy Beckett, Raquel Godoy-Ruiz, and David J. Weber
- Subjects
Models, Molecular ,0301 basic medicine ,Protein Conformation ,Protein subunit ,Bacterial Toxins ,030106 microbiology ,Enterotoxin ,Crystallography, X-Ray ,medicine.disease_cause ,Corrections ,Biophysical Phenomena ,Enterotoxins ,03 medical and health sciences ,Bacterial Proteins ,Protein Domains ,Chlorocebus aethiops ,medicine ,Animals ,structural biology ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Vero Cells ,X-ray crystallography ,ADP Ribose Transferases ,Pore-forming toxin ,Binding Sites ,Multidisciplinary ,Clostridioides difficile ,Toxin ,Chemistry ,Cryoelectron Microscopy ,Clostridium difficile ,Biological Sciences ,NMR ,3. Good health ,Macromolecular assembly ,Biophysics and Computational Biology ,030104 developmental biology ,Structural biology ,Biochemistry ,cryo-EM - Abstract
Significance There is a burden from Clostridium difficile infection throughout the world, and the Centers for Disease Control reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin is associated with the most serious outbreaks of drug-resistant C. difficile infection in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of the C. difficile binary toxin, termed CDTb. Surprisingly, 2 structures were solved from a single sample that help explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods., Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.
- Published
- 2020
10. Retrieving functional pathways of biomolecules from single-particle snapshots
- Author
-
Ghoncheh Mashayekhi, Amedee des Georges, Mrinal Shekhar, Ali Dashti, Abbas Ourmazd, Abhishek Singharoy, Salah Salah, Joachim Frank, Peter Schwander, and Danya Ben Hail
- Subjects
0301 basic medicine ,Macromolecular Substances ,Science ,Allosteric regulation ,Molecular Conformation ,General Physics and Astronomy ,Sequence (biology) ,Computational biology ,Molecular Dynamics Simulation ,Ligands ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Molecular dynamics ,Computational biophysics ,Computational models ,Binding site ,lcsh:Science ,chemistry.chemical_classification ,Multidisciplinary ,Binding Sites ,030102 biochemistry & molecular biology ,Ryanodine receptor ,Biomolecule ,Cryoelectron Microscopy ,Ryanodine Receptor Calcium Release Channel ,General Chemistry ,Calcium Channel Agonists ,030104 developmental biology ,Structural biology ,chemistry ,lcsh:Q ,Function (biology) - Abstract
A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, we present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Our approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations., There is a great interest in retrieving functional pathways from cryo-EM single-particle data. Here, the authors present an approach that combines cryo-EM with advanced data-analytical methods and molecular dynamics simulations to reveal the functional pathways traversed on experimentally derived energy landscapes using the ryanodine receptor type 1 as an example.
- Published
- 2020
11. Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex
- Author
-
Jan Steyaert, Søren Heissel, Thomas J. Cahill, Tara C. Marcink, Li-Yin Huang, Oliver B. Clarke, Robert J. Lefkowitz, Sarah Triest, Chuan Hong, Rick Huang, Anthony H. Nguyen, Henrik Molina, Yong Zi Tan, Fadi Samaan, John Little, Alex R.B. Thomsen, Amedee des Georges, Xin Chen, Danya Ben-Hail, Ali Masoudi, Venkata P. Dandey, Roger K. Sunahara, Jacob P. Mahoney, Zhiheng Yu, Department of Bio-engineering Sciences, and Structural Biology Brussels
- Subjects
Models, Molecular ,Receptors, Vasopressin ,G protein ,Endosome ,Protein Conformation ,Biophysics ,beta-2 ,Endosomes ,Medical and Health Sciences ,Article ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,G-Protein-Coupled ,0302 clinical medicine ,Protein structure ,Structural Biology ,GTP-Binding Proteins ,Models ,Receptors ,Arrestin ,Animals ,Humans ,Receptor ,Molecular Biology ,beta-Arrestins ,030304 developmental biology ,G protein-coupled receptor ,0303 health sciences ,Chemistry ,Cryoelectron Microscopy ,Molecular ,Biological Sciences ,Cell biology ,Adrenergic ,Chemical Sciences ,Phosphorylation ,Cattle ,Receptors, Adrenergic, beta-2 ,Generic health relevance ,Signal transduction ,030217 neurology & neurosurgery ,Vasopressin ,Signal Transduction ,Developmental Biology - Abstract
Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR–G protein–β-arr ‘megaplex’. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β 2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (β 2V 2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.
- Published
- 2019
12. Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly
- Author
-
Heather M. Neu, Christopher Peralta, Paul T. Wilder, Braden M. Roth, Sianny Christanti, Danya Ben-Hail, Amedee des Georges, Mary E. Cook, Sarah L. J. Michel, Richard R. Rustandi, Xingjian Xu, Edwin Pozharski, Catherine Lancaster, Thomas E. Cleveland, John W. Loughney, Jessica W. Olson, Alexander Grishaev, David J. Weber, Raquel Godoy-Ruiz, Dorothy Beckett, Alex D. MacKerell, Kaylin A. Adipietro, Wenbo Yu, Kristen M. Varney, and Adam Kristopeit
- Subjects
0303 health sciences ,Pore-forming toxin ,030306 microbiology ,Chemistry ,Toxin ,Protein subunit ,Computational biology ,Enterotoxin ,Clostridium difficile ,medicine.disease_cause ,3. Good health ,Macromolecular assembly ,03 medical and health sciences ,Structural biology ,medicine ,Binding site ,030304 developmental biology - Abstract
TargetingClostridium difficileinfection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed theC. difficiletoxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures foractivated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). ForAsymCDTb, a novel Ca2+binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI.SIGNIFICANCE STATEMENTThere is a high burden fromC. difficileinfection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a thirdC. difficilebinary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21stcentury. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings ofC. difficiletoxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.
- Published
- 2019
- Full Text
- View/download PDF
13. Author response: Mechanisms of opening and closing of the bacterial replicative helicase
- Author
-
Paul Db Olinares, Alex J. Noble, Kelly R. Molloy, Danaya Pakotiprapha, Amedee des Georges, Brian T. Chait, David Jeruzalmi, Andrew Catalano, Jillian Chase, Martin A. Samuels, and Edward T. Eng
- Subjects
media_common.quotation_subject ,Closing (real estate) ,biology.protein ,Helicase ,Biology ,media_common ,Cell biology - Published
- 2018
14. ENRICH: a fast method to improve the quality of flexible macromolecular reconstructions
- Author
-
Javier Vargas, Mohsen Kazemi, Carlos Oscar S. Sorzano, José María Carazo, and Amedee des Georges
- Subjects
Flexibility (engineering) ,0303 health sciences ,Computer science ,030303 biophysics ,Resolution (electron density) ,Optical flow ,Single particle analysis ,law.invention ,03 medical and health sciences ,Quality (physics) ,law ,Microscopy ,Particle ,Electron microscope ,Projection (set theory) ,Algorithm ,030304 developmental biology - Abstract
Cryo-electron microscopy using single particle analysis requires the computational averaging of thousands of projection images captured from identical macromolecules. However, macromolecules usually present some degree of flexibility showing different conformations. Computational approaches are then required to classify heterogeneous single particle images into homogeneous sets corresponding to different structural states. Nonetheless, sometimes the attainable resolution of reconstructions obtained from these smaller homogeneous sets is compromised because of reduced number of particles or lack of images at certain macromolecular orientations. In these situations, the current solution to improve map resolution is returning to the electron microscope and collect more data. In this work, we present a fast approach to partially overcome this limitation for heterogeneous data sets. Our method is based on deforming and then moving particles between different conformations using an optical flow approach. Particles are then merged into a unique conformation obtaining reconstructions with improved resolution, contrast and signal-to-noise ratio, then, partially circumventing many issues that impact obtaining high quality reconstructions from small data sets. We present experimental results that show clear improvements in the quality of obtained 3D maps, however, there are also limits to this approach, which we discuss in the manuscript.
- Published
- 2018
- Full Text
- View/download PDF
15. Viral Entry Properties Required for Fitness in Humans Are Lost through Rapid Genomic Change during Viral Isolation
- Author
-
Marion Ferren, Negar Makhsous, Dolly B. Aquino, Ryan C. Shean, Matteo Porotto, Sho Iketani, Anne Moscona, Alexander L. Greninger, Bert K. Rima, Amedee des Georges, Cyrille Mathieu, Immunobiologie des infections virales – Immunobiology of Viral Infections (IbIV), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Iketani, Sho, Shean, Ryan C., Ferren, Marion, Makhsous, Negar, Aquino, Dolly B., Georges, Amedee de, Rima, Bert, Mathieu, Cyrille, Porotto, Matteo, Moscona, Anne, and Greninger, Alexander L.
- Subjects
0301 basic medicine ,Nonsynonymous substitution ,Virus Cultivation ,Entry mechanism ,parainfluenza virus ,030106 microbiology ,DNA Mutational Analysis ,entry mechanisms ,Adaptation, Biological ,viral fitness ,Paramyxoviru ,Genome, Viral ,Biology ,Microbiology ,Respirovirus Infections ,Virus ,03 medical and health sciences ,Metagenomic ,viral evolution ,paramyxovirus ,Viral entry ,Virology ,Humans ,Serial Passage ,Gene ,ComputingMilieux_MISCELLANEOUS ,metagenomics ,lung infection ,Viral fitne ,Virus Internalization ,Fusion protein ,Phenotype ,QR1-502 ,3. Good health ,Parainfluenza Virus 3, Human ,Human Parainfluenza Virus ,030104 developmental biology ,Viral evolution ,Mutation ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,Genetic Fitness ,viral fusion ,Parainfluenza viru ,Research Article - Abstract
IMPORTANCE Human parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions., Human parainfluenza viruses cause a large burden of human respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus 3 (HPIV-3) evolves in culture. Cultured viruses differ in their properties compared to clinical strains. We present a genome-wide survey of HPIV-3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). Nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV-3 primary culture isolates and 23 HPIV-3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN’s dimerization/F-interaction site, a site critical for activation of viral fusion. Alterations in HN dimer interface residues known to favor infection in culture occurred within 4 days (H552 and N556). A novel cluster of residues at a different face of the HN dimer interface emerged (P241 and R242) and imply a role in HPIV-3-mediated fusion. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV-3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity. These results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the HN-F complex required for fitness by circulating viruses.
- Published
- 2018
- Full Text
- View/download PDF
16. Author response: Routine single particle CryoEM sample and grid characterization by tomography
- Author
-
Peter D. Kwong, William J. Rice, Clinton S. Potter, Lawrence Shapiro, Anchi Cheng, Priyamvada Acharya, Venkata P. Dandey, Hui Wei, Micah Rapp, Alex J. Noble, Giovanna Scapin, David Jeruzalmi, Jillian Chase, Amedee des Georges, Zhening Zhang, Laura Y. Kim, Yong Zi Tan, Julia Brasch, Bridget Carragher, Edward T. Eng, and Carl J. Negro
- Subjects
Materials science ,Optics ,business.industry ,Particle ,Tomography ,business ,Grid ,Sample (graphics) ,Characterization (materials science) - Published
- 2018
17. Engineered ferritin for lanthanide binding
- Author
-
Amedee des Georges, Claudia Testi, Maciej Białasek, Magdalena Król, Alberto Boffi, Tomasz P. Rygiel, Paola Baiocco, Matilde Cardoso Trabuco, Bartłomiej Taciak, Linda Celeste Montemiglio, Irene Benni, Lorenzo Calisti, Andrea Ilari, and Alessandra Bonamore
- Subjects
0301 basic medicine ,Fluorescence-lifetime imaging microscopy ,Luminescence ,lcsh:Medicine ,Plasma protein binding ,Protein Engineering ,01 natural sciences ,Lanthanoid Series Elements ,Fluorophotometry ,Mice ,Spectrum Analysis Techniques ,Aromatic Amino Acids ,Neoplasms ,Fluorescence Resonance Energy Transfer ,Electron Microscopy ,Amino Acids ,lcsh:Science ,Microscopy ,Multidisciplinary ,Crystallography ,biology ,Chemistry ,Organic Compounds ,Physics ,Electromagnetic Radiation ,genetics and molecular biology (all) ,agricultural and biological sciences (all) ,Tryptophan ,Condensed Matter Physics ,Fluorescence ,Spectrophotometry ,Physical Sciences ,Crystal Structure ,Research Article ,Chemical Elements ,Protein Binding ,Iron ,chemistry.chemical_element ,Terbium ,010402 general chemistry ,Research and Analysis Methods ,03 medical and health sciences ,Cell Line, Tumor ,Escherichia coli ,Solid State Physics ,Animals ,Humans ,biochemistry ,Binding site ,Ferritin ,Binding Sites ,lcsh:R ,Organic Chemistry ,Chemical Compounds ,Biology and Life Sciences ,Proteins ,Protein Complexes ,Electron Cryo-Microscopy ,Protein engineering ,0104 chemical sciences ,030104 developmental biology ,Förster resonance energy transfer ,Apoferritins ,Biophysics ,biology.protein ,lcsh:Q - Abstract
Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 A and cryo-EM at 7 A resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295–375 nm, fluorescence excitation.
- Published
- 2018
18. Ryanodine Receptor Structure and Function in Health and Disease
- Author
-
Andrew R. Marks, Joachim Frank, Gaetano Santulli, Amedee des Georges, Daniel R. Lewis, Santulli, Gaetano, Lewis, Daniel, des Georges, Amedee, Marks, Andrew R., and Frank, Joachim
- Subjects
0301 basic medicine ,Gating ,Neurotransmission ,Synaptic Transmission ,Article ,Muscular Dystrophies ,Calcium in biology ,03 medical and health sciences ,0302 clinical medicine ,Diabetes Mellitus ,medicine ,Animals ,Humans ,Calcium Signaling ,Muscular dystrophy ,Ion channel ,Ryanodine receptor ,Chemistry ,Endoplasmic reticulum ,Skeletal muscle ,Neurodegenerative Diseases ,Ryanodine Receptor Calcium Release Channel ,musculoskeletal system ,medicine.disease ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Mutation ,Calcium ,030217 neurology & neurosurgery - Abstract
Ryanodine receptors (RyRs) are ubiquitous intracellular calcium (Ca2+) release channels required for the function of many organs including heart and skeletal muscle, synaptic transmission in the brain, pancreatic beta cell function, and vascular tone. In disease, defective function of RyRs due either to stress (hyperadrenergic and/or oxidative overload) or genetic mutations can render the channels leaky to Ca2+ and promote defective disease-causing signals as observed in heat failure, muscular dystrophy, diabetes mellitus, and neurodegerative disease. RyRs are massive structures comprising the largest known ion channel-bearing macromolecular complex and exceeding 3 million Daltons in molecular weight. RyRs mediate the rapid release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) to stimulate cellular functions through Ca2+-dependent processes. Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the determination of atomic-level structures for RyR for the first time. These structures have illuminated the mechanisms by which these critical ion channels function and interact with regulatory ligands. In the present chapter we discuss the structure, functional elements, gating and activation mechanisms of RyRs in normal and disease states.
- Published
- 2018
19. Routine Single Particle CryoEM Sample and Grid Characterization by Tomography
- Author
-
Laura Y. Kim, Bridget Carragher, Edward T. Eng, Anchi Cheng, Giovanna Scapin, Lawrence Shapiro, Priyamvada Acharya, Julia Brasch, Alex J. Noble, Amedee des Georges, Venkata P. Dandey, Hui Wei, Micah Rapp, Carl J. Negro, Jillian Chase, Yong Zi Tan, William J. Rice, Zhening Zhang, Clinton S. Potter, David Jeruzalmi, and Peter D. Kwong
- Subjects
0301 basic medicine ,Electron Microscope Tomography ,genetic structures ,Structural Biology and Molecular Biophysics ,protomo ,02 engineering and technology ,Biochemistry ,Fructose-Bisphosphate Aldolase ,Microscopy ,Biology (General) ,0303 health sciences ,air-water ,Orientation (computer vision) ,fiducial-less ,Air ,General Neuroscience ,General Medicine ,single particle ,021001 nanoscience & nanotechnology ,Grid ,Sample (graphics) ,Tools and Resources ,Characterization (materials science) ,Ice thickness ,Particles ,Medicine ,Rabbits ,Tomography ,cryoET ,0210 nano-technology ,DnaB Helicases ,Sugar Alcohol Dehydrogenases ,Proteasome Endopeptidase Complex ,Materials science ,QH301-705.5 ,Surface Properties ,Science ,Biophysics ,tomography ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,None ,Electron microscopy ,Escherichia coli ,Animals ,030304 developmental biology ,General Immunology and Microbiology ,Cryoelectron Microscopy ,Water ,Computational physics ,030104 developmental biology ,Apoferritins ,Particle - Abstract
Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.
- Published
- 2017
- Full Text
- View/download PDF
20. Structure of mammalian eIF3 in the context of the 43S preinitiation complex
- Author
-
Christopher U.T. Hellen, Vidya Dhote, Lauriane Kuhn, Tatyana V. Pestova, Yaser Hashem, Amedee des Georges, and Joachim Frank
- Subjects
Models, Molecular ,RNA, Transfer, Met ,Eukaryotic Initiation Factor-3 ,Protein subunit ,Eukaryotic Initiation Factor-2 ,Codon, Initiator ,Biology ,Bioinformatics ,Ribosome ,Protein Structure, Secondary ,Article ,Eukaryotic translation ,Peptide Initiation Factors ,Humans ,Initiation factor ,Eukaryotic Small Ribosomal Subunit ,RNA, Messenger ,COP9 signalosome ,Peptide Chain Initiation, Translational ,Ribosome Subunits, Small, Eukaryotic ,Binding Sites ,Multidisciplinary ,Cryoelectron Microscopy ,Cell biology ,Protein Subunits ,Ribosome Subunits ,Multiprotein Complexes ,Transfer RNA ,Ribosomes ,RNA Helicases - Abstract
During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5'-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Å resolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.
- Published
- 2015
21. Structure of a mammalian ryanodine receptor
- Author
-
Joachim Frank, Wayne A. Hendrickson, Ran Zalk, Oliver B. Clarke, Filippo Mancia, Andrew R. Marks, Amedee des Georges, Steven Reiken, and Robert A. Grassucci
- Subjects
Gating ,Biology ,Article ,Tacrolimus Binding Proteins ,03 medical and health sciences ,Cytosol ,0302 clinical medicine ,medicine ,Animals ,Muscle, Skeletal ,Ion channel ,030304 developmental biology ,RYR1 ,0303 health sciences ,Multidisciplinary ,Voltage-dependent calcium channel ,Ryanodine receptor ,Cell Membrane ,Cryoelectron Microscopy ,Skeletal muscle ,Ryanodine Receptor Calcium Release Channel ,musculoskeletal system ,Protein Structure, Tertiary ,Transmembrane domain ,medicine.anatomical_structure ,Biochemistry ,Biophysics ,Calcium ,Rabbits ,medicine.symptom ,Ion Channel Gating ,030217 neurology & neurosurgery ,Muscle contraction - Abstract
Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation–contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 A. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+. Using electron cryomicroscopy, the closed-state structure of rabbit RyR1 is determined at 4.8 A resolution; analysis confirms that the RyR1 architecture consists of a six-transmembrane ion channel with a cytosolic α-solenoid scaffold, and suggests a mechanism for Ca2+-induced channel opening. Muscle contraction is regulated by the concentration of calcium ions in the cytoplasm of muscle cells. Ryanodine receptors (RyR) release Ca2+ from the sarcoplasmic reticulum to induce muscle contraction. Dysfunction of these channels contributes to the pathophysiology of important human diseases including muscular dystrophy. Three papers in this issue of Nature report high-resolution electron cryomicroscopy structures of the 2.2 MDa ryanodine receptor RyR1. Efremov et al. report the structure of rabbit RyR1 at 8.5 A resolution the presence of Ca2+ in a 'partly open' state, and at 6.1 A resolution in the absence of Ca2+ in a closed state. Zalk et al. report the rabbit RyR1 structure at 4.8 A in the absence of Ca2+ in a closed state. And third, Yan et al. report the structure of rabbit RyR1 bound to its modulator FKBP12 at a near-atomic resolution of 3.8 A. These papers reveal how calcium binding to the EF-hand domain of RyR1 regulates channel opening and facilitates calcium-induced calcium release. The authors also note that disease-causing mutations are clustered in regions of the channel that appear to be critical for normal channel function.
- Published
- 2014
22. Conformational Dynamics and Energy Landscapes of Ligand Binding in RyR1
- Author
-
Abbas Ourmazd, Joachim Frank, Peter Schwander, Ghoncheh Mashayekhi, Ali Dashti, Amedee des Georges, and Danya Ben Hail
- Subjects
RYR1 ,0303 health sciences ,Ryanodine receptor ,030303 biophysics ,Skeletal muscle ,chemistry.chemical_element ,Calcium ,03 medical and health sciences ,medicine.anatomical_structure ,Biochemistry ,chemistry ,medicine ,Biophysics ,Binding site ,030304 developmental biology ,Binding domain - Abstract
Using experimental single-particle cryo-EM snapshots of ryanodine receptor (RyR1), a Ca2+-channel involved in skeletal muscle excitation/contraction coupling, we present quantitative free-energy landscapes, reaction coordinates, and three-dimensional movies of the continuous conformational changes associated with the binding of activating ligands. Our results show multiple routes to ligand binding with comparable branching ratios. All high-probability routes involve significant conformational changes before and after the binding of ligands. We also present new insights into the local structural changes along the ligand-binding route, including accommodations at the calcium, ATP, and caffeine binding sites. These observations shed new light on the mechanisms and conformational routes to ligand binding.
- Published
- 2017
23. CryoET of Single Particle CryoEM Grids Reveals Widespread Particle Adsorption to the Air-Water Interface, Which May be Reduced with New Plunging Techniques
- Author
-
Giovanna Scapin, Jillian Chase, Hui Wei, Venkata P. Dandey, Lawrence Shapiro, Priyamvada Acharya, William J. Rice, Clinton S. Potter, David Jeruzalmi, Bridget Carragher, Laura Y. Kim, Zhening Zhang, Kwong, Edward T. Eng, Carl J. Negro, Amedee des Georges, Micah Rapp, Anchi Cheng, Alex J. Noble, Yong Zi Tan, and Julia Brasch
- Subjects
010302 applied physics ,Materials science ,Chemical physics ,Air water interface ,0103 physical sciences ,Particle adsorption ,Particle ,02 engineering and technology ,021001 nanoscience & nanotechnology ,0210 nano-technology ,01 natural sciences ,Instrumentation - Published
- 2018
24. Mechanisms of opening and closing of the bacterial replicative helicase
- Author
-
Brian T. Chait, Jillian Chase, Amedee des Georges, Kelly R. Molloy, David Jeruzalmi, Edward T. Eng, Danaya Pakotiprapha, Alex J. Noble, Martin A. Samuels, Paul Db Olinares, and Andrew Catalano
- Subjects
0301 basic medicine ,Models, Molecular ,Protein Conformation ,Structural Biology and Molecular Biophysics ,02 engineering and technology ,01 natural sciences ,Biochemistry ,chemistry.chemical_compound ,Structural Biology ,General Materials Science ,Biology (General) ,Closing (morphology) ,replication initiation ,media_common ,cryogenic electron microscopy ,biology ,Chemistry ,General Neuroscience ,General Medicine ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Bacteriophage lambda ,Cell biology ,Medicine ,0210 nano-technology ,DnaB Helicases ,Research Article ,Protein Binding ,QH301-705.5 ,Protein subunit ,Science ,media_common.quotation_subject ,DNA, Single-Stranded ,DNA replication ,010403 inorganic & nuclear chemistry ,helicase loader ,General Biochemistry, Genetics and Molecular Biology ,Inorganic Chemistry ,03 medical and health sciences ,Viral Proteins ,Biochemistry and Chemical Biology ,Escherichia coli ,Physical and Theoretical Chemistry ,dnaB helicase ,General Immunology and Microbiology ,Cryoelectron Microscopy ,Closing (real estate) ,E. coli ,Helicase ,0104 chemical sciences ,030104 developmental biology ,Structural biology ,Replication Initiation ,Biophysics ,biology.protein ,DnaB replicative helicase ,DNA - Abstract
Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the Escherichia coli DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.
- Published
- 2019
25. Structure of the mammalian ribosomal pre-termination complex associated with eRF1•eRF3•GDPNP
- Author
-
Joachim Frank, Robert A. Grassucci, Christopher U.T. Hellen, Derek J. Taylor, Amedee des Georges, Tatyana V. Pestova, Anett Unbehaun, and Yaser Hashem
- Subjects
Models, Molecular ,GTPase ,Peptide Chain Termination, Translational ,Ribosomal RNA ,Biology ,Stop codon ,GTP Phosphohydrolases ,3. Good health ,Eukaryotic translation ,Biochemistry ,Start codon ,Structural Biology ,Codon usage bias ,Codon, Terminator ,Genetics ,Humans ,Guanosine Triphosphate ,Ribosomes ,Peptide Termination Factors - Abstract
Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3’s GTPase activity.
- Published
- 2013
26. Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome
- Author
-
Deborah F. Kelly, Joachim Frank, Robert Langlois, Amedee des Georges, Robert A. Grassucci, Cheri M. Hampton, Sanchaita Das, Gyanesh Sharma, and Jesper Pallesen
- Subjects
Models, Molecular ,Ribosome Subunits, Small, Eukaryotic ,Protein Conformation ,Cryoelectron Microscopy ,Receptors, Cell Surface ,Translation (biology) ,Biology ,Receptors for Activated C Kinase ,Ribosome ,Article ,Neoplasm Proteins ,Cell biology ,GTP-binding protein regulators ,Protein structure ,GTP-Binding Proteins ,Structural Biology ,Ribosome Subunits ,Protein Biosynthesis ,Humans ,Eukaryotic Small Ribosomal Subunit ,Protein Kinase C ,Protein kinase C - Abstract
Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC βII binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map which shows that PKC βII interacts with RACK1, a seven-bladed β-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1. This study is a first step in understanding the molecular framework of PKC βII/RACK1 interaction and its role in translation.
- Published
- 2013
27. Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus
- Author
-
Amedee des Georges, Iwan A. T. Schaap, Claudia Veigel, and Frederic Eghiaian
- Subjects
Light ,viruses ,Lipid Bilayers ,Biophysics ,Hemagglutinin (influenza) ,Electrons ,Microscopy, Atomic Force ,Biochemistry ,Capsid ,Viral Envelope Proteins ,Viral envelope ,Scattering, Radiation ,Particle Size ,Lipid bilayer ,Molecular Biology ,Micelles ,Liposome ,biology ,Chemistry ,Bilayer ,Cryoelectron Microscopy ,virus diseases ,Cell Biology ,Hydrogen-Ion Concentration ,Orthomyxoviridae ,Lipids ,Kinetics ,Crystallography ,Membrane protein ,Liposomes ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Stress, Mechanical ,Neuraminidase ,Membrane biophysics ,Molecular Biophysics - Abstract
The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm(-1)) than a viral protein-capsid coat and sustains deformations of one-third of the virion's diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.
- Published
- 2012
28. Improving 3D reconstructions of macromolecular conformations
- Author
-
Carlos Oscar S. Sorzano, José María Carazo, Moshen Kazemi, Amedee des Georges, and Javier Vargas
- Subjects
Inorganic Chemistry ,Structural Biology ,Biophysics ,General Materials Science ,Physical and Theoretical Chemistry ,Condensed Matter Physics ,Biochemistry - Published
- 2018
29. Mal3, the Schizosaccharomyces pombe homolog of EB1, changes the microtubule lattice
- Author
-
Linda A. Amos, Douglas Robert Drummond, Miho Katsuki, Michael Osei, Amedee des Georges, and Robert A. Cross
- Subjects
Models, Molecular ,biology ,Microtubule-associated protein ,Cryoelectron Microscopy ,macromolecular substances ,Plasma protein binding ,biology.organism_classification ,Microtubules ,Article ,Cell biology ,Tubulin ,Structural Biology ,Microtubule ,Schizosaccharomyces ,Schizosaccharomyces pombe ,biology.protein ,Amino Acid Sequence ,Schizosaccharomyces pombe Proteins ,Microtubule-Associated Proteins ,Molecular Biology ,Peptide sequence ,Gene Deletion ,Protein Binding ,Microtubule nucleation - Abstract
In vitro studies of pure tubulin have suggested that tubulin heterodimers in cells assemble into B-lattice microtubules, where the 8-nm dimers in adjacent protofilaments are staggered by 0.9 nm. This arrangement requires the tube to close by forming a seam with an A-lattice, in which the protofilaments are staggered by 4.9 nm. Here we show that Mal3, an EB1 family tip-tracking protein, drives tubulin to assemble in vitro into exclusively 13-protofilament microtubules with a high proportion of A-lattice protofilament contacts. We present a three-dimensional cryo-EM reconstruction of a purely A-lattice microtubule decorated with Mal3, in which Mal3 occupies the groove between protofilaments and associates closely with one tubulin monomer. We propose that Mal3 promotes assembly by binding to freshly formed tubulin polymer and particularly favors any with A-lattice arrangement. These results reopen the question of microtubule structure in cells.
- Published
- 2008
30. Functional Study of the Ryanodine Receptor Type 1 using Cryo-Electron Microscopy
- Author
-
Oliver B. Clarke, Amedee des Georges, Qi Yuan, Wayne A. Hendrickson, Ran Zalk, Andrew R. Marks, Robert A. Grassucci, Joachim Frank, and Kendall J. Condon
- Subjects
RYR1 ,Membrane protein ,Structural biology ,Biochemistry ,Cryo-electron microscopy ,Ryanodine receptor ,Chemistry ,Biophysics ,Gating ,Binding site ,Calcium in biology - Abstract
Cryo-electron microscopy (cryo-EM) is revolutionizing the structural biology of membrane proteins. It has allowed the structural study of long sought after targets, such as the type 1 ryanodine receptor (RyR1). But another very important advantage is that it allows to study the complex dynamics associated with membrane protein function.The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. We used single-particle cryo-EM to study the dynamics of RyR1 in multiple functional states, revealing the conformational changes key to channel gating and ligand-dependent activation. The binding sites for the channel activators Ca2+, ATP and caffeine were identified and the conformational changes associated with their binding observed independently. They induce by themselves a priming of the cytoplasmic assembly without pore dilation. In contrast, in the presence of all three activating ligands, open and closed states of the pore were obtained from the same sample, enabling analysis of conformational changes associated with gating. The analysis of multiple functional states was greatly facilitated by the use of holey-gold grids, which allowed cryo-EM reconstructions to reach high resolution with much fewer number of particles than with conventional holey carbon grids.
- Published
- 2017
31. Discovery of neutralizing SARS-CoV-2 antibodies enriched in a unique antigen specific B cell cluster.
- Author
-
Stine Sofie Frank Lende, Nanna Møller Barnkob, Randi Westh Hansen, Harsh Bansia, Mike Vestergaard, Frederik Holm Rothemejer, Anne Worsaae, Deijona Brown, Maria Lange Pedersen, Anna Halling Folkmar Rahimic, Anna Karina Juhl, Torben Gjetting, Lars Østergaard, Amédée Des Georges, Laurent-Michel Vuillard, Mariane Høgsbjerg Schleimann, Klaus Koefoed, and Martin Tolstrup
- Subjects
Medicine ,Science - Abstract
Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.
- Published
- 2023
- Full Text
- View/download PDF
32. High-resolution Cryo-EM Structure of the Trypanosoma brucei Ribosome: A Case Study
- Author
-
Yaser Hashem, Joachim Frank, Susan Madison-Antenucci, Jie Fu, Qin Zhang, Hstau Y. Liao, Amedee des Georges, Sarah N. Buss, Eric Westhof, Chandrajit L. Bajaj, Robert A. Grassucci, Amy Jobe, Fabrice Jossinet, and Robert Langlois
- Subjects
Physics ,Data processing ,Contrast transfer function ,Cryo-electron microscopy ,3D reconstruction ,Microscopy ,Resolution (electron density) ,Spatial frequency ,Overfitting ,Biological system - Abstract
Single-particle cryo-electron microscopy has the immense advantage over crystallography in being able to image frozen-hydrated biological complexes in their “native” state, in solution. For years the ribosome has been the benchmark sample for particles without symmetry. It has witnessed steady improvement in resolution from the very first single-particle 3D reconstruction to today’s reconstructions at near-atomic resolution. In this study, we describe the different steps of sample preparation, data collection, data processing, and modeling that led to the 5A structure of the T. brucei ribosome [32]. A local resolution estimation demonstrates the extent to which resolution can be anisotropic and pinpoints regions of higher heterogeneity or structural flexibility. This study also shows an example of misuse of spatial frequency filters leading to overfitting of the data and the artifacts that can be observed in the resulting density map.
- Published
- 2013
33. Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit
- Author
-
Robert A. Grassucci, Robert Langlois, Joachim Frank, Hstau Y. Liao, Vidya Dhote, Christopher U.T. Hellen, Amedee des Georges, Tatyana V. Pestova, and Yaser Hashem
- Subjects
Genetics ,Models, Molecular ,Ribosome Subunits, Small, Eukaryotic ,Multidisciplinary ,Eukaryotic Initiation Factor-3 ,fungi ,Cryoelectron Microscopy ,Biology ,Regulatory Sequences, Ribonucleic Acid ,Ribosome ,Binding, Competitive ,Internal ribosome entry site ,Ribosome Subunits ,Classical Swine Fever Virus ,Eukaryotic initiation factor ,Protein Biosynthesis ,Transfer RNA ,Initiation factor ,P-site ,Animals ,Humans ,RNA, Viral ,Eukaryotic Small Ribosomal Subunit ,Rabbits ,Ribosomes - Abstract
Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5-7, 9-12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.
- Published
- 2013
34. Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29
- Author
-
Vidya Dhote, Hstau Y. Liao, Robert A. Grassucci, Joachim Frank, Amedee des Georges, Tatyana V. Pestova, Christopher U.T. Hellen, Yaser Hashem, and Robert Langlois
- Subjects
Models, Molecular ,Eukaryotic Initiation Factor-2 ,Molecular Sequence Data ,Biophysics ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Eukaryotic initiation factor ,RNA, Ribosomal, 18S ,Initiation factor ,Animals ,Humans ,Eukaryotic Small Ribosomal Subunit ,Peptide Chain Initiation, Translational ,030304 developmental biology ,Mammals ,0303 health sciences ,Base Sequence ,Cell-Free System ,Eukaryotic Large Ribosomal Subunit ,Biochemistry, Genetics and Molecular Biology(all) ,030302 biochemistry & molecular biology ,Cryoelectron Microscopy ,Molecular biology ,Cell biology ,EIF1 ,Ribonucleoproteins ,RNA, Ribosomal ,Transcription preinitiation complex ,Rabbits ,Eukaryotic Ribosome ,030217 neurology & neurosurgery ,RNA Helicases - Abstract
Summary Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNA i Met ), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6 A resolution. It reveals that eIF2 interacts with the 40S subunit via its α subunit and supports Met-tRNA i Met in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.
- Published
- 2013
35. High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome
- Author
-
Joachim Frank, Fabrice Jossinet, Qin Zhang, Jie Fu, Yaser Hashem, Susan Madison-Antenucci, Amy Jobe, Hstau Y. Liao, Robert A. Grassucci, Eric Westhof, Sarah N. Buss, Chandrajit L. Bajaj, and Amedee des Georges
- Subjects
Models, Molecular ,Multidisciplinary ,Eukaryotic Large Ribosomal Subunit ,5.8S ribosomal RNA ,Cryoelectron Microscopy ,Trypanosoma brucei brucei ,Molecular Conformation ,Computational biology ,Ribosomal RNA ,Biology ,Molecular biology ,Models, Biological ,Article ,Ribosomal protein ,RNA, Ribosomal ,Large ribosomal subunit ,Protein Biosynthesis ,Yeasts ,Eukaryotic Small Ribosomal Subunit ,Eukaryotic Ribosome ,Ribosomes ,RNA, Protozoan ,50S - Abstract
Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.
- Published
- 2013
36. Structural Basis for Gating and Activation of RyR1
- Author
-
Joachim Frank, Kendall J. Condon, Robert A. Grassucci, Oliver B. Clarke, Wayne A. Hendrickson, Qi Yuan, Ran Zalk, Amedee des Georges, and Andrew R. Marks
- Subjects
0301 basic medicine ,Protein domain ,Gating ,Biology ,Ligands ,Bioinformatics ,General Biochemistry, Genetics and Molecular Biology ,Calcium in biology ,Tacrolimus Binding Proteins ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Caffeine ,medicine ,Animals ,Binding site ,RYR1 ,Binding Sites ,Ryanodine receptor ,Cryoelectron Microscopy ,Ryanodine Receptor Calcium Release Channel ,Calcium Channel Agonists ,Transmembrane domain ,030104 developmental biology ,Biophysics ,Calcium ,Rabbits ,sense organs ,medicine.symptom ,Ion Channel Gating ,030217 neurology & neurosurgery ,Muscle Contraction ,Muscle contraction - Abstract
The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.
- Published
- 2016
37. 33 High-resolution cryo-EM structure of theTrypanosoma brucei80S: a unique eukaryotic ribosome
- Author
-
Sarah N. Buss, Joachim Frank, Jie Fu, Fabrice Jossinet, Qin Zhang, Yaser Hashem, Hstau Y. Liao, Amy Jobe, Susan Madison-Antenucci, Amedee des Georges, Bob Grassucci, Eric Westhof, and Chandrajit L. Bajaj
- Subjects
Genetics ,biology ,Eukaryotic Large Ribosomal Subunit ,General Medicine ,Ribosomal RNA ,Trypanosoma brucei ,biology.organism_classification ,Ribosome ,18S ribosomal RNA ,Structural Biology ,Eukaryotic initiation factor ,parasitic diseases ,Eukaryotic Small Ribosomal Subunit ,Eukaryotic Ribosome ,Molecular Biology - Abstract
Eukaryotic 80S ribosomes of known structure are far more complex than their 70S bacterial counterparts. Those from Saccharomyces cerevisiae, Tetrahymena thermophila, and Triticum aestivum, for example, bear insertions of ribosomal RNA (rRNA) called expansion segments (ES) and additional ribosomal proteins. The ribosomes of the kinetoplastid Trypanosoma brucei, though, are especially fascinating: structurally and their other kinetoplastids’ ribosomes bear very large ESs, as well as smaller ESs, and protein extensions. Additionally, T. brucei ribosomes require novel protein factors for maturation, although they do not require several eukaryotic initiation factors or a recycling factor. As a species, T. brucei is fascinating not only in terms of structure, but also in terms of gene expression and even public health: the species is responsible for the incurable, terminal human African Trypanosomiasis (sleeping sickness); and during post-transcriptional regulation, a single common RNA segment called a splice l...
- Published
- 2013
38. Cryo-EM structure of the human ferritin–transferrin receptor 1 complex
- Author
-
Linda Celeste Montemiglio, Claudia Testi, Pierpaolo Ceci, Elisabetta Falvo, Martina Pitea, Carmelinda Savino, Alessandro Arcovito, Giovanna Peruzzi, Paola Baiocco, Filippo Mancia, Alberto Boffi, Amédée des Georges, and Beatrice Vallone
- Subjects
Science - Abstract
The human transferrin receptor 1 (CD71) is a transmembrane protein responsible for iron uptake. Here the authors present the 3.9 Å resolution cryo-EM structure of the CD71 ectodomain-human ferritin (H-Ft) complex and find that H-Ft binds a CD71 region different from the transferrin one that overlaps with the surface recognized by select pathogens.
- Published
- 2019
- Full Text
- View/download PDF
39. The Influenza Virus Mechanical Properties Are Dominated By Its Lipid Envelope
- Author
-
Claudia Veigel, Amedee des Georges, Iwan A. T. Schaap, Frederic Eghiaian, and John J. Skehel
- Subjects
0303 health sciences ,Viral matrix protein ,viruses ,M1 protein ,Biophysics ,Lipid bilayer fusion ,Viral membrane ,Biology ,01 natural sciences ,Virology ,Virus ,3. Good health ,03 medical and health sciences ,Viral envelope ,Viral entry ,0103 physical sciences ,biology.protein ,010306 general physics ,Lipid bilayer ,030304 developmental biology - Abstract
The influenza (flu) virus causes yearly epidemics, and has claimed the life of tens of millions of people in the last century. Flu viruses need to travel from one host to a new one, where they inject their RNA genome by a membrane fusion mechanism. Before fusion the flu virus RNA genome protected by an envelope made of a matrix protein (M1) layer surrounded by a lipid membrane. The exact role of M1 protein and its mode of interaction with the viral membrane are unknown.We have set out to investigate the mechanical design of influenza virus: we imaged and characterized mechanical properties of influenza virions by atomic force microscopy (AFM). We compared the response of the viral particle with the behavior of simplified model systems to understand the role of the various parts of the viral structure in its mechanical properties.Influenza virions proved to be very soft compared to the other “protein-enveloped” viruses that have been characterized by AFM so far. The stiffness of viral particles was comparable to that of similar-sized small unilamellar lipid vesicles and virosomes. Our results suggest that the M1 protein does not mechanically reinforce the flu virus envelope and that M1 may not directly interact with the inner side of the viral membrane. Hypothesis on the conditions under which influenza virus will persist during transmission will be discussed.
- Published
- 2009
40. Engineered ferritin for lanthanide binding.
- Author
-
Lorenzo Calisti, Matilde Cardoso Trabuco, Alberto Boffi, Claudia Testi, Linda Celeste Montemiglio, Amédée des Georges, Irene Benni, Andrea Ilari, Bartłomiej Taciak, Maciej Białasek, Tomasz Rygiel, Magdalena Król, Paola Baiocco, and Alessandra Bonamore
- Subjects
Medicine ,Science - Abstract
Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295-375 nm, fluorescence excitation.
- Published
- 2018
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.