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The single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins (NPs) and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a folded RNA and a deformed vRNP associated with a looped RNA. Interestingly, the looped RNA was a double-stranded RNA, which likely comprises a nascent RNA and the template RNA detached from NPs of the vRNP. These results suggest that while some vRNPs keep their helical structures during RNA synthesis, for the repeated cycle of RNA synthesis, others accidentally become structurally deformed, which likely results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural feature of vRNPs during RNA synthesis.
Masahiro Nakano; Yukihiko Sugita; Noriyuki Kodera; Sho Miyamoto; Yukiko Muramoto; Matthias Wolf; Takeshi Noda. Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis. Communications Biology 2021, 4, 1 -10.
AMA StyleMasahiro Nakano, Yukihiko Sugita, Noriyuki Kodera, Sho Miyamoto, Yukiko Muramoto, Matthias Wolf, Takeshi Noda. Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis. Communications Biology. 2021; 4 (1):1-10.
Chicago/Turabian StyleMasahiro Nakano; Yukihiko Sugita; Noriyuki Kodera; Sho Miyamoto; Yukiko Muramoto; Matthias Wolf; Takeshi Noda. 2021. "Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis." Communications Biology 4, no. 1: 1-10.
During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. We found that high serum antibody titers can persist for more than 9 months post infection. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.
Melissa M. Matthews; Tae Gyun Kim; Satoshi Shibata; Noriko Shibata; Christian Butcher; Jaekyung Hyun; Keon Young Kim; Theodore Robb; Siang Sheng Jheng; Masashi Narita; Tomoari Mori; Mary Collins; Matthias Wolf. COVID-19 serological survey using micro blood sampling. Scientific Reports 2021, 11, 1 -9.
AMA StyleMelissa M. Matthews, Tae Gyun Kim, Satoshi Shibata, Noriko Shibata, Christian Butcher, Jaekyung Hyun, Keon Young Kim, Theodore Robb, Siang Sheng Jheng, Masashi Narita, Tomoari Mori, Mary Collins, Matthias Wolf. COVID-19 serological survey using micro blood sampling. Scientific Reports. 2021; 11 (1):1-9.
Chicago/Turabian StyleMelissa M. Matthews; Tae Gyun Kim; Satoshi Shibata; Noriko Shibata; Christian Butcher; Jaekyung Hyun; Keon Young Kim; Theodore Robb; Siang Sheng Jheng; Masashi Narita; Tomoari Mori; Mary Collins; Matthias Wolf. 2021. "COVID-19 serological survey using micro blood sampling." Scientific Reports 11, no. 1: 1-9.
Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.
Nadishka Jayawardena; Linde Miles; Laura Burga; Charles Rudin; Matthias Wolf; John Poirier; Mihnea Bostina. N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection. Viruses 2021, 13, 769 .
AMA StyleNadishka Jayawardena, Linde Miles, Laura Burga, Charles Rudin, Matthias Wolf, John Poirier, Mihnea Bostina. N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection. Viruses. 2021; 13 (5):769.
Chicago/Turabian StyleNadishka Jayawardena; Linde Miles; Laura Burga; Charles Rudin; Matthias Wolf; John Poirier; Mihnea Bostina. 2021. "N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection." Viruses 13, no. 5: 769.
During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol (1, 2) in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. We found that high serum antibody titers can persist for at least up to 6.5 months post infection. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.
Melissa M. Matthews; Tae Gyun Kim; Satoshi Shibata; Noriko Shibata; Christian Butcher; Jaekyung Hyun; Keon Young Kim; Theodore Robb; Siang Sheng Jheng; Masashi Narita; Tomoari Mori; Mary Collins; Matthias Wolf. COVID-19 serological survey using micro blood sampling. 2020, 1 .
AMA StyleMelissa M. Matthews, Tae Gyun Kim, Satoshi Shibata, Noriko Shibata, Christian Butcher, Jaekyung Hyun, Keon Young Kim, Theodore Robb, Siang Sheng Jheng, Masashi Narita, Tomoari Mori, Mary Collins, Matthias Wolf. COVID-19 serological survey using micro blood sampling. . 2020; ():1.
Chicago/Turabian StyleMelissa M. Matthews; Tae Gyun Kim; Satoshi Shibata; Noriko Shibata; Christian Butcher; Jaekyung Hyun; Keon Young Kim; Theodore Robb; Siang Sheng Jheng; Masashi Narita; Tomoari Mori; Mary Collins; Matthias Wolf. 2020. "COVID-19 serological survey using micro blood sampling." , no. : 1.
Bacterial adhesion is a general strategy for host–microbe and microbe–microbe interactions. Adhesive pili are essential for colonization, biofilm formation, virulence and pathogenesis of many environmental and pathogenic bacteria1,2. Members of the class Bacteroidia have unique type V pili, assembled by protease-mediated polymerization3. Porphyromonas gingivalis is the main contributor to periodontal disease and its type V pili are a key factor for its virulence4. However, the structure of the polymerized pilus and its assembly mechanism are unknown. Here we show structures of polymerized and monomeric states of FimA stalk pilin from P. gingivalis, determined by cryo-electron microscopy and crystallography. The atomic model of assembled FimA shows that the C-terminal strand of a donor subunit is inserted into a groove in the β-sheet of an acceptor subunit after N-terminal cleavage by the protease RgpB. The C terminus of the donor strand is essential for polymerization. We propose that type V pili assemble via a sequential polar assembly mechanism at the cell surface, involving protease-mediated strand exchange, employed by various Gram-negative species belonging to the class Bacteroidia. Our results reveal functional surfaces related to pathogenic properties of polymerized FimA. These insights may facilitate development of antibacterial drugs.
Satoshi Shibata; Mikio Shoji; Kodai Okada; Hideyuki Matsunami; Melissa M. Matthews; Katsumi Imada; Koji Nakayama; Matthias Wolf. Structure of polymerized type V pilin reveals assembly mechanism involving protease-mediated strand exchange. Nature Microbiology 2020, 5, 830 -837.
AMA StyleSatoshi Shibata, Mikio Shoji, Kodai Okada, Hideyuki Matsunami, Melissa M. Matthews, Katsumi Imada, Koji Nakayama, Matthias Wolf. Structure of polymerized type V pilin reveals assembly mechanism involving protease-mediated strand exchange. Nature Microbiology. 2020; 5 (6):830-837.
Chicago/Turabian StyleSatoshi Shibata; Mikio Shoji; Kodai Okada; Hideyuki Matsunami; Melissa M. Matthews; Katsumi Imada; Koji Nakayama; Matthias Wolf. 2020. "Structure of polymerized type V pilin reveals assembly mechanism involving protease-mediated strand exchange." Nature Microbiology 5, no. 6: 830-837.
The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.
Yoshimasa Takizawa; Cheng-Han Ho; Hiroaki Tachiwana; Hideyuki Matsunami; Wataru Kobayashi; Midori Suzuki; Yasuhiro Arimura; Tetsuya Hori; Tatsuo Fukagawa; Melanie D. Ohi; Matthias Wolf; Hitoshi Kurumizaka. Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome. Structure 2019, 28, 44 -53.e4.
AMA StyleYoshimasa Takizawa, Cheng-Han Ho, Hiroaki Tachiwana, Hideyuki Matsunami, Wataru Kobayashi, Midori Suzuki, Yasuhiro Arimura, Tetsuya Hori, Tatsuo Fukagawa, Melanie D. Ohi, Matthias Wolf, Hitoshi Kurumizaka. Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome. Structure. 2019; 28 (1):44-53.e4.
Chicago/Turabian StyleYoshimasa Takizawa; Cheng-Han Ho; Hiroaki Tachiwana; Hideyuki Matsunami; Wataru Kobayashi; Midori Suzuki; Yasuhiro Arimura; Tetsuya Hori; Tatsuo Fukagawa; Melanie D. Ohi; Matthias Wolf; Hitoshi Kurumizaka. 2019. "Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome." Structure 28, no. 1: 44-53.e4.
Bacterial locomotion by rotating flagella is achieved through the hook, which transmits torque from the motor to the filament. The hook is a tubular structure composed of a single type of protein, yet it adopts a curved shape. To perform its function, it must be simultaneously flexible and torsionally rigid. The molecular mechanism by which chemically identical subunits form such a dynamic structure is unknown. Here, we show the complete structure of the hook from Salmonella enterica in its supercoiled ‘curved’ state, at 2.9 Å resolution. Subunits in the curved hook are grouped into 11 distinctive conformations, each shared along 11 protofilaments. The domains of the elongated hook subunit behave as rigid bodies connected by two hinge regions. The reconstituted model demonstrates how identical subunits can dynamically change conformation by physical interactions while bending. These multiple subunit states contradict the two-state model, which is a key feature of flagellar polymorphism.
Satoshi Shibata; Hideyuki Matsunami; Shin-Ichi Aizawa; Matthias Wolf. Torque transmission mechanism of the curved bacterial flagellar hook revealed by cryo-EM. Nature Structural & Molecular Biology 2019, 26, 941 -945.
AMA StyleSatoshi Shibata, Hideyuki Matsunami, Shin-Ichi Aizawa, Matthias Wolf. Torque transmission mechanism of the curved bacterial flagellar hook revealed by cryo-EM. Nature Structural & Molecular Biology. 2019; 26 (10):941-945.
Chicago/Turabian StyleSatoshi Shibata; Hideyuki Matsunami; Shin-Ichi Aizawa; Matthias Wolf. 2019. "Torque transmission mechanism of the curved bacterial flagellar hook revealed by cryo-EM." Nature Structural & Molecular Biology 26, no. 10: 941-945.
Many archaea swim by means of archaella. While the archaellum is similar in function to its bacterial counterpart, its structure, composition, and evolution are fundamentally different. Archaella are related to archaeal and bacterial type IV pili. Despite recent advances, our understanding of molecular processes governing archaellum assembly and stability is still incomplete. Here, we determine the structures of Methanococcus archaella by X‐ray crystallography and cryo‐EM. The crystal structure of Methanocaldococcus jannaschii FlaB1 is the first and only crystal structure of any archaellin to date at a resolution of 1.5 Å, which is put into biological context by a cryo‐EM reconstruction from Methanococcus maripaludis archaella at 4 Å resolution created with helical single‐particle analysis. Our results indicate that the archaellum is predominantly composed of FlaB1. We identify N‐linked glycosylation by cryo‐EM and mass spectrometry. The crystal structure reveals a highly conserved metal‐binding site, which is validated by mass spectrometry and electron energy‐loss spectroscopy. We show in vitro that the metal‐binding site, which appears to be a widespread property of archaellin, is required for filament integrity.
Vladimir A Meshcheryakov; Satoshi Shibata; Makoto Tokoro Schreiber; Alejandro Villar‐Briones; Kenneth F Jarrell; Shin‐Ichi Aizawa; Matthias Wolf. High‐resolution archaellum structure reveals a conserved metal‐binding site. EMBO reports 2019, 20, e46340 .
AMA StyleVladimir A Meshcheryakov, Satoshi Shibata, Makoto Tokoro Schreiber, Alejandro Villar‐Briones, Kenneth F Jarrell, Shin‐Ichi Aizawa, Matthias Wolf. High‐resolution archaellum structure reveals a conserved metal‐binding site. EMBO reports. 2019; 20 (5):e46340.
Chicago/Turabian StyleVladimir A Meshcheryakov; Satoshi Shibata; Makoto Tokoro Schreiber; Alejandro Villar‐Briones; Kenneth F Jarrell; Shin‐Ichi Aizawa; Matthias Wolf. 2019. "High‐resolution archaellum structure reveals a conserved metal‐binding site." EMBO reports 20, no. 5: e46340.
Recently, the use of oncolytic viruses in cancer therapy has become a realistic therapeutic option. Seneca Valley Virus (SVV) is a newly discovered picornavirus, which has earned a significant reputation as a potent oncolytic agent. Anthrax toxin receptor 1 (ANTXR1), one of the cellular receptors for the protective antigen secreted by Bacillus anthracis, has been identified as the high-affinity cellular receptor for SVV. Here, we report the structure of the SVV-ANTXR1 complex determined by single-particle cryo-electron microscopy analysis at near-atomic resolution. This is an example of a shared receptor structure between a mammalian virus and a bacterial toxin. Our structure shows that ANTXR1 decorates the outer surface of the SVV capsid and interacts with the surface-exposed BC loop and loop II of VP1, “the puff” of VP2 and “the knob” of VP3. Comparison of the receptor-bound capsid structure with the native capsid structure reveals that receptor binding induces minor conformational changes in SVV capsid structure, suggesting the role of ANTXR1 as an attachment receptor. Furthermore, our results demonstrate that the capsid footprint on the receptor is not conserved in anthrax toxin receptor 2 (ANTXR2), thereby providing a molecular mechanism for explaining the exquisite selectivity of SVV for ANTXR1.
Nadishka Jayawardena; Laura N. Burga; Richard A. Easingwood; Yoshimasa Takizawa; Matthias Wolf; Mihnea Bostina. Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus. Proceedings of the National Academy of Sciences 2018, 115, E10934 -E10940.
AMA StyleNadishka Jayawardena, Laura N. Burga, Richard A. Easingwood, Yoshimasa Takizawa, Matthias Wolf, Mihnea Bostina. Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus. Proceedings of the National Academy of Sciences. 2018; 115 (46):E10934-E10940.
Chicago/Turabian StyleNadishka Jayawardena; Laura N. Burga; Richard A. Easingwood; Yoshimasa Takizawa; Matthias Wolf; Mihnea Bostina. 2018. "Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus." Proceedings of the National Academy of Sciences 115, no. 46: E10934-E10940.
Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical nucleoprotein–RNA complex, VP24, VP30, VP35 and viral polymerase1,3. The nucleoprotein–RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion4,5. RNA binding and nucleoprotein oligomerization are synergistic and do not readily occur independently6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core4,5, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola virus nucleoprotein–RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development.
Yukihiko Sugita; Hideyuki Matsunami; Yoshihiro Kawaoka; Takeshi Noda; Matthias Wolf. Cryo-EM structure of the Ebola virus nucleoprotein–RNA complex at 3.6 Å resolution. Nature 2018, 563, 137 -140.
AMA StyleYukihiko Sugita, Hideyuki Matsunami, Yoshihiro Kawaoka, Takeshi Noda, Matthias Wolf. Cryo-EM structure of the Ebola virus nucleoprotein–RNA complex at 3.6 Å resolution. Nature. 2018; 563 (7729):137-140.
Chicago/Turabian StyleYukihiko Sugita; Hideyuki Matsunami; Yoshihiro Kawaoka; Takeshi Noda; Matthias Wolf. 2018. "Cryo-EM structure of the Ebola virus nucleoprotein–RNA complex at 3.6 Å resolution." Nature 563, no. 7729: 137-140.
Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the ALB1 gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The ALB1 enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the ALB1 enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the ALB1 enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the ALB1 enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the ALB1 enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the ALB1 enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone–DNA contacts may be responsible for the enhanced DNA accessibility in the nucleosome with the ALB1 enhancer DNA.
Yoshimasa Takizawa; Hiroki Tanaka; Shinichi Machida; Masako Koyama; Kazumitsu Maehara; Yasuyuki Ohkawa; Paul Wade; Matthias Wolf; Hitoshi Kurumizaka. Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence. Open Biology 2018, 8, 1 .
AMA StyleYoshimasa Takizawa, Hiroki Tanaka, Shinichi Machida, Masako Koyama, Kazumitsu Maehara, Yasuyuki Ohkawa, Paul Wade, Matthias Wolf, Hitoshi Kurumizaka. Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence. Open Biology. 2018; 8 (3):1.
Chicago/Turabian StyleYoshimasa Takizawa; Hiroki Tanaka; Shinichi Machida; Masako Koyama; Kazumitsu Maehara; Yasuyuki Ohkawa; Paul Wade; Matthias Wolf; Hitoshi Kurumizaka. 2018. "Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence." Open Biology 8, no. 3: 1.
Heterochromatin plays important roles in transcriptional silencing and genome maintenance by the formation of condensed chromatin structures, which determine the epigenetic status of eukaryotic cells. The trimethylation of histone H3 lysine 9 (H3K9me3), a target of heterochromatin protein 1 (HP1), is a hallmark of heterochromatin formation. However, the mechanism by which HP1 folds chromatin-containing H3K9me3 into a higher-order structure has not been elucidated. Here we report the three-dimensional structure of the H3K9me3-containing dinucleosomes complexed with human HP1α, HP1β, and HP1γ, determined by cryogenic electron microscopy with a Volta phase plate. In the structures, two H3K9me3 nucleosomes are bridged by a symmetric HP1 dimer. Surprisingly, the linker DNA between the nucleosomes does not directly interact with HP1, thus allowing nucleosome remodeling by the ATP-utilizing chromatin assembly and remodeling factor (ACF). The structure depicts the fundamental architecture of heterochromatin.
Shinichi Machida; Yoshimasa Takizawa; Masakazu Ishimaru; Yukihiko Sugita; Satoshi Sekine; Jun-Ichi Nakayama; Matthias Wolf; Hitoshi Kurumizaka. Structural Basis of Heterochromatin Formation by Human HP1. Molecular Cell 2018, 69, 385 -397.e8.
AMA StyleShinichi Machida, Yoshimasa Takizawa, Masakazu Ishimaru, Yukihiko Sugita, Satoshi Sekine, Jun-Ichi Nakayama, Matthias Wolf, Hitoshi Kurumizaka. Structural Basis of Heterochromatin Formation by Human HP1. Molecular Cell. 2018; 69 (3):385-397.e8.
Chicago/Turabian StyleShinichi Machida; Yoshimasa Takizawa; Masakazu Ishimaru; Yukihiko Sugita; Satoshi Sekine; Jun-Ichi Nakayama; Matthias Wolf; Hitoshi Kurumizaka. 2018. "Structural Basis of Heterochromatin Formation by Human HP1." Molecular Cell 69, no. 3: 385-397.e8.
Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30 years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules.We provide an overview of the cryo-EM method and introduce contemporary studies to investigate biomolecular structure and dynamics, including examples from the recent literature.Cryo-EM is a powerful tool for the investigation of biological macromolecular structures including analysis of their dynamics by using advanced image-processing algorithms. The method has become even more widely applicable with present-day single particle analysis and electron tomography.The cryo-EM method can be used to determine the three-dimensional structure of biomacromolecules in near native condition at close to atomic resolution, and has the potential to reveal conformations of dynamic molecular complexes. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.
Kazuyoshi Murata; Matthias Wolf. Cryo-electron microscopy for structural analysis of dynamic biological macromolecules. Biochimica et Biophysica Acta (BBA) - General Subjects 2018, 1862, 324 -334.
AMA StyleKazuyoshi Murata, Matthias Wolf. Cryo-electron microscopy for structural analysis of dynamic biological macromolecules. Biochimica et Biophysica Acta (BBA) - General Subjects. 2018; 1862 (2):324-334.
Chicago/Turabian StyleKazuyoshi Murata; Matthias Wolf. 2018. "Cryo-electron microscopy for structural analysis of dynamic biological macromolecules." Biochimica et Biophysica Acta (BBA) - General Subjects 1862, no. 2: 324-334.
Since the development of parallel electron energy loss spectroscopy (EELS), charge-coupled devices (CCDs) have been the default detectors for EELS. With the recent development of electron-counting direct-detection cameras, micrographs can be acquired under very low electron doses at significantly improved signal-to-noise ratio. In spectroscopy, in particular in combination with a monochromator, the signal can be extremely weak and the detection limit is principally defined by noise introduced by the detector. Here we report the use of an electron-counting direct-detection camera for EEL spectroscopy. We studied the oxygen K edge of amorphous ice and obtained a signal noise ratio up to 10 times higher than with a conventional CCD. We report the application of electron counting to record time-resolved EEL spectra of a biological protein embedded in amorphous ice, revealing chemical changes observed in situ while exposed by the electron beam. A change in the fine structure of nitrogen K and the carbon K edges were recorded during irradiation. A concentration of 3 at% nitrogen was detected with a total electron dose of only 1.7 e−/Å2, extending the boundaries of EELS signal detection at low electron doses.
Alan Maigné; Matthias Wolf. Low-dose electron energy-loss spectroscopy using electron counting direct detectors. Microscopy 2017, 67, i86 -i97.
AMA StyleAlan Maigné, Matthias Wolf. Low-dose electron energy-loss spectroscopy using electron counting direct detectors. Microscopy. 2017; 67 (suppl_1):i86-i97.
Chicago/Turabian StyleAlan Maigné; Matthias Wolf. 2017. "Low-dose electron energy-loss spectroscopy using electron counting direct detectors." Microscopy 67, no. suppl_1: i86-i97.
Radiation damage is a major limiting factor in obtaining high resolution images in cryo-electron microscopy. Quantifying and understanding those radiation damages are considerable challenges. While it seems commonly admitted that the major part of the damages comes from radiolysis[1], local charging within the ice or the sample has been mentioned several time as a source of damage[2].Due to the isolator behavior of frozen H2O, samples undergo electrostatic charging. While it has never been directly measured, this charging effect is often observable in cryo-electron microscopy in the form of beam deflection or Coulomb-explosion (as shown in Figure 1).Aranova et al. showed that Electron Energy Loss Spectroscopy (EELS) could be used to measure the production of H2 and O2 associated to “bubbles” often observed in cryo-EM when damages start to occurs [3]. A very interesting finding in their report is that no bubbles were observed in pure frozen water (without sample in it). One possible explanation is that radicals recombine more quickly in H2O than in frozen hydrated specimens in a Transmission Electron Microsocope.During this presentation, we will study with mono-chromated EELS the effects of the incident electron beam on frozen H2O as well as on frozen aqueous samples. Both the radiolysis and the electrostatic aspects of the beam/matter interaction can be measured with EELS using Kramer-Kronig formula (through dielectric formalism) and Valence EELS (to look at chemical structure changes and bubbles production). We will show that depending on the type of electron illumination (STEM or TEM), the nature and intensity of those damages can vary. We will, then, look at several possible solutions to reduce such damages.Keywords:EELS;Cryo Electron Microscopy;Monochromator;Live Science;Dielectric Fucntion;Beam damages
Alan Maigne; Matthias Wolf. EELS analysis of the interaction betweeen frozen acqueous samples and incident electrons in TEM. European Microscopy Congress 2016: Proceedings 2016, 877 -878.
AMA StyleAlan Maigne, Matthias Wolf. EELS analysis of the interaction betweeen frozen acqueous samples and incident electrons in TEM. European Microscopy Congress 2016: Proceedings. 2016; ():877-878.
Chicago/Turabian StyleAlan Maigne; Matthias Wolf. 2016. "EELS analysis of the interaction betweeen frozen acqueous samples and incident electrons in TEM." European Microscopy Congress 2016: Proceedings , no. : 877-878.
Archaeal flagella are unique structures that share functional similarity with bacterial flagella, but are structurally related to bacterial type IV pili. The flagellar accessory protein FlaH is one of the conserved components of the archaeal motility system. However, its function is not clearly understood. Here, we present the 2.2 Å resolution crystal structure of FlaH from the hyperthermophilic archaeon, Methanocaldococcus jannaschii. The protein has a characteristic RecA-like fold, which has been found previously both in archaea and bacteria. We show that FlaH binds to immobilized ATP – however, it lacks ATPase activity. Surface plasmon resonance analysis demonstrates that ATP affects the interaction between FlaH and the archaeal motor protein FlaI. In the presence of ATP, the FlaH-FlaI interaction becomes significantly weaker. A database search revealed similarity between FlaH and several DNA-binding proteins of the RecA superfamily. The closest structural homologs of FlaH are KaiC-like proteins, which are archaeal homologs of the circadian clock protein KaiC from cyanobacteria. We propose that one of the functions of FlaH may be the regulation of archaeal motor complex assembly. This article is protected by copyright. All rights reserved.
Vladimir A. Meshcheryakov; Matthias Wolf. Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly. Protein Science 2016, 25, 1147 -1155.
AMA StyleVladimir A. Meshcheryakov, Matthias Wolf. Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly. Protein Science. 2016; 25 (6):1147-1155.
Chicago/Turabian StyleVladimir A. Meshcheryakov; Matthias Wolf. 2016. "Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly." Protein Science 25, no. 6: 1147-1155.
The flagellar accessory protein FlaH is thought to be one of the essential components of an archaeal motility system. However, to date biochemical and structural information about this protein has been limited. Here, the crystallization of FlaH from the hyperthermophilic archaeon Methanocaldococcus jannaschii is reported. Protein crystals were obtained by the vapour-diffusion method. These crystals belonged to space group P3₁21, with unit-cell parameters a=b=131.42, c=89.35 Å. The initial solution of the FlaH structure has been determined by multiple-wavelength anomalous dispersion phasing using a selenomethionine-derivatized crystal.
Vladimir A. Meshcheryakov; Young-Ho Yoon; Hideyuki Matsunami; Matthias Wolf. Purification, crystallization and preliminary X-ray crystallographic analysis of the flagellar accessory protein FlaH from the methanogenic archaeon Methanocaldococcus jannaschii. Acta Crystallographica Section F Structural Biology Communications 2014, 70, 1543 -5.
AMA StyleVladimir A. Meshcheryakov, Young-Ho Yoon, Hideyuki Matsunami, Matthias Wolf. Purification, crystallization and preliminary X-ray crystallographic analysis of the flagellar accessory protein FlaH from the methanogenic archaeon Methanocaldococcus jannaschii. Acta Crystallographica Section F Structural Biology Communications. 2014; 70 (11):1543-5.
Chicago/Turabian StyleVladimir A. Meshcheryakov; Young-Ho Yoon; Hideyuki Matsunami; Matthias Wolf. 2014. "Purification, crystallization and preliminary X-ray crystallographic analysis of the flagellar accessory protein FlaH from the methanogenic archaeon Methanocaldococcus jannaschii." Acta Crystallographica Section F Structural Biology Communications 70, no. 11: 1543-5.