Reserch overviews

We investigate structures of biomolecules, biomacromolecular complexes, and cellular architecture using electron microscopy, and reveal a relationship between the structure and the function.

The Highlights:

 

A structure of Melbournevirus was investigated by cryo-EM, showing T=304 capsid

Okamoto K, Miyazaki N, Reddy HKN., Hantke MF, Maia FRNC., Larsson DSD, Abergel C, Claverie JM, Hajdu J, Murata K, & Svenda M (2018) Cryo-EM structure of a Marseilleviridae virus particle reveals a large internal microassembly. Virology, 516, 239-245. doi:10.1016/j.virol.2018.01.021

A model of schizophrenia and intellectual disability, "Schnurri-2 knockout mouse" was investigated by SBF-SEM, showing premature morphological features in the hippocampal dentate gyrus of the brain.

Nakao A, Miyazaki N, Ohira K, Hagihara H, Takagi T, Usuda N, Ishii S, Murata K, & Miyakawa T (2017) Immature morphological properties in subcellular-scale structures in the dentate gyrus of Schnurri-2 knockout mice: a model for schizophrenia and intellectual disability. Mol Brain, 10:60.

Order of the molecules in chitinase crystal was determined by cryo - electron microscope tomography, which made possible the quantitative measurement of reactive element process of chitinase.

Nakamura A, Tasaki T, Okuni Y, Song C, Murata K, Kozai T, Hara M, Sugimoto H, Suzuki K, Watanabe T, Uchihashi T, Noji H, & Iino R (2017) Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis. Phys Chem Chem Phys, doi: 10.1039/c7cp04606e

The detailed structure of the largest virus "Pithovirus" was investigated by cryo-electron microscopes.

Okamoto K, Miyazaki N, Song C, Maia FRNC, Reddy HKN, Abergel C, Claverie J-M, Hajdu J, Svenda M, & Murata K (2017) Structural variability and complexity of the giant Pithovirus sibericum particle revealed by high-voltage electron cryo-tomography and energy-filtered electron cryo-microscopy. Sci Rep, 7, 13291.

The flexible structure of UGGT, a enzyme for protein quality control, was visualized by cryo-EM.

Satoh T, Song C, Zhu T, Toshimori T, Murata K, Hayashi Y, Kamikubo H, Uchihashi T, & Kato K (2017) Visualisation of a flexible modular structure of the ER folding-sensor enzyme UGGT. Sci Rep, 7:12142

A review of cryo-EM for investigating dynamic protein structures.

Murata K & Wolf M (2017) Cryo-electron microscopy for structural analysis of dynamic biological macromolecules. Biochim Biophys Acta, 1862(2), 324-334.

Diversity of structures across genera in the calicivirus family was investigated by cryo-EM.

Conley M, Emmott E, Orton R, Taylor D, Carneiro DG, Murata K, Goodfellow IG, Hansman GS, & Bhella D (2017) Vesivirus 2117 capsids more closely resemble sapovirus and lagovirus particles than other known vesivirus structures. J Gen Virol,98: 68–76.

SBF-SEM revealed that afadin molecule plays an important role in morphogenesis of mouse hippocampal mossy fiber synapse.

Sai K, Wang S, Kaito A, Fujiwara T, Maruo T, Itoh Y, Miyata M, Sakakibara S, Miyazaki N, Murata K, Yamaguchi Y, Haruta T, Nishioka H, Motojima Y, Komura M, Kimura K, Mandai K, Takai Y, & Mizoguchi A (2017) Multiple roles of afadin in the ultrastructural morphogenesis of mouse hippocampal mossy fiber synapses. J Comp Neurol, 525: 2719–2734.

High voltage TEM revealed that the PV membrane formed by the symbiotic chlorella with the host 's paramecium is firmly interacting with the host mitochondria via a structure of wedge.

Song C, Murata K, & Suzaki T (2017) Intracellular symbiosis of algae with possible involvement of mitochondrial dynamics. Sci Rep, 7: 1221.

The moment when cyanophage P-SSP7 adsorbs to and infects the host cyanobacteria was visualied by cryo-electron tomography.

Murata K, Zhang Q, Gerardo Galaz-Montoya J, Fu C, Coleman ML, Osburne MS, Schmid MF, Sullivan MB, Chisholm SW, & Chiu W (2017) Visualizing Adsorption of Cyanophage P-SSP7 onto Marine Prochlorococcus. Sci Rep, 7: 44176.

Hydrogel microparticles are expected to be applied to drug delivery carriers. The hydrated structure of the hydrogel microparticles was visualized by cryo-EM.

Watanabe T, Kobayashi C, Song C, Murata K, Kureha T, Suzuki D (2016) Impact of Spatial Distribution of Charged Groups in Core Poly( N isopropylacrylamide)-Based Microgels on the Resultant Composite Structures Prepared by Seeded Emulsion Polymerization of Styrene. Langmuir, 32:12760–12773.

We discovered that cyanobacteria produce a structure similar to chromosomes when cell division, and this structure was revealed by high voltage cryo-EM.

Murata K, Hagiwara S, Kimori Y, & Kaneko Y (2016) Ultrastructure of compacted DNA in cyanobacteria by high-voltage cryo-electron tomography.Sci Rep, 6: 34934.

The functional receptor of mouse norovirus was identified as a proteins CD300lf and CD300ld, and the binding site of the receptors were simulated on our cryo-EM map.

Haga K, Fujimoto A, Takai-Todaka R , Miki M, Doan YH, Murakami K, Yokoyama M, Murata K, Nakanishi A, & Katayama K. (2016) Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells. Proc Natl Acad Sci USA, 113(41), E6248–E6255.

Structure of toti-like virus, Omotoriver virus (OmRV) was investigated by cryo-EM. It was suggested that the long protrusion structure, which was considered necessary for invasion into the host cell from the atmosphere, is not necessary, and the raised structure of the surface of the viral particle directly relates to this.

Okamoto K, Miyazaki N, Larsson DS, Kobayashi D, Svenda M, Mühlig K, Maia FR, Gunn LH, Isawa H, Kobayashi M, Sawabe K, Murata K, & Hajdu J. (2016) The infectious particle of insectborne totivirus-like Omono River virus has raised ridges and lacks fibre complexes. Sci Rep, 6: 33170.

We found a new mechanism to determine the direction of cell division in the ascidian embryo. As a result of the SBF-SEM observation, it was found that the structure extending from the adjacent cell determines this.

Negishi T, Miyazaki N, Murata K, Yasuo H, & Ueno N. (2016) Physical association between a novel plasma-membrane structure and centrosome orients cell division. Elife, 5, e16550.

3D structure of a spiral microorganism discovered in the see with a depth of 1,200 m was investigated by high voltage TEM, showing it contains a small number of organelles as a characteristic of extreme environment.

Yamaguchi M, Yamada H, Higuchi K, Yamamoto Y, Arai S, Murata K, Mori Y, Furukawa H, Uddin MS & Chibana H (2016) High-voltage electron microscopy tomography and structome analysis of unique spiral bacteria from the deep sea. Microscopy, 65, 363-369.

3D structure of podocytes in the glomerulus of the kidney was reconstructed using SBF-SEM and its detailed formation process was elucidated.

Ichimura K, Kakuta S, Kawasaki Y, Miyaki T, Nonami T, Miyazaki N, Nakao T, Enomoto S, Arai S, Koike M, Murata K, & Sakai T. (2017) Morphological process of podocyte development revealed by block- face scanning electron microscopy. J Cell Sci, 130, 132–142.

Hydrated structure of polystylen microgels prepared by seedied emulsion polymerization of stylene was ingvestigated by electron cryomicroscpy. Polystylene particles were located on the surface of the microgel.

Kobayashi C, Watanabe T, Murata K, Kureha T, & Suzuki D. (2016). Localization of Polystyrene Particles on the Surface of Poly(N-isopropylacrylamide-co-methacrylic acid) Microgels Prepared by Seeded Emulsion Polymerization of Styrene. Langmuir, 32(6): 1429-1439.

The structure of innexin gap junction ion channel was investigated by ceyo-electron microscopy. It consisted of hexadecameric larger size rings than those of connexin.

Oshima A, Matsuzawa T, Murata K, Tani K, & Fujiyoshi Y. (2016). Hexadecameric structure of an invertebrate gap junction channel. J Mol Biol, 428(6): 1227-1236.

The structual developement in human maralia parasite was investigated by SBF-SEM, suggesting a new mechianism to transport materials between the Erythrocyte membrane and the parasite.

Sakaguchi M, Miyazaki N, Fujioka H, Kaneko O, & Murata K.(2016). hree-dimensional analysis of morphological changes in the malaria parasite infected red blood cell by serial block-face scanning electron microscopy. J Struct Biol, 193(3): 162-171.

We investigated the dedailed structures of legs in tanaid crustacean using SBF-SEM, wehre spindles were formed from the tip of the legs by mixing materials from two spearate tubes.

Kaji T, Kakui K, Miyazaki N, Murata K, & A R Palmer (2016). Mesoscale morphology at nanoscale resolution: serial block-face scanning electron microscopy reveals fine 3D detail of a novel silk spinneret system in a tube-building tanaid crustacean. Front Zool, 13:14.

We revealed the relationsip between microtubles (MTs) and microfilaments (MFs) in plant cells using electron tomgoraphy. MFs make a croslink between MTs forming a bundle of MTs durint the formation of pre-proface bands.

Takeuchi M, Karahara I, Kajimura N, Takaoka A, Murata K, Misaki K, Yonemura S, Staehelin LA & Mineyuki Y. (2016). Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells. Mol Biol Cell, 27(11):
1809-1820.

We revealed the fist 3D structure of sapovirus, where there was a large gap between outer and inner capsids. The inner capsid also showed a lot of epitops.

Miyazaki N, Taylor D W, Hansman G S, & Murata K. (2015). Antigenic and cryo-electron microscopy structure analysis of a chimeric sapovirus capsid. J Virol, 90(5): 2664-2675.

     

We developed a new method for HVEM tomography to analyze the structure of synapse connections in neural network, wherer the immuno histochemistry is combined with the OTO en bloc staining. The method makes a possible to efficiently find and analyze the structure of the taeget synapse.

Satoh K, Takanami K, Murata K, Kawata M, Sakamoto T, & Sakamoto H. (2015). Effective synaptome analysis of itch-mediating neurons in the spinal cord : A novel immunohistochemical methodology using high-voltage electron microscopy. Neurosci Let 599 : 86–91.

A mutant of budding yeast lakcking Cdc48p ATPase shows unusual mitochondrial morphology. We investigate it using SBF-SEM. The mitochondria in the mutant present their flagmentation and aggregation.

Miyazaki N, Esaki M, Ogura T, & Murata K. (2014). Serial block-face scanning electron microscopy for three-dimensional analysis of morphological changes in mitochondria regulated by Cdc48p/p97 ATPase. J Struct Biol, 187: 187-93.

A whole cell structure of a budding yeast was investigated using high-voltage electron tomography. Especially, the bright field STEM revealed the clear 3D structures and their localizations of major organella, nuleus, mitochondoria, valuoles in the cell.

Murata K, Esaki M, Ogura T, Arai S, Yamamoto Y, & Tanaka N. (2014). Whole-Cell Imaging of the Budding Yeast Saccharomyces cerevisiae by High-Voltage Scanning Transmission Electron Tomography. Ultramicroscopy, 146: 39-45.

A homolog of the self-assemby factor in 20S protease functions as the activation factor in Archaea. Electron tomography revealed the binding feature of the homolog with 20S protease.

Kumoi K, Satoh T, Murata K, Hiromoto T, Mizushima T, Kamiya Y, Noda M, Uchiyama S, Yagi H & Kato K. (2013). An archaeal homolog of proteasome assembly factor functions as a proteasome activator. PloS one 8: e60294.

Identified the binding site of the monoclonal aintibody recognizing the capsid domain inside of the norovirus capsid

Hansman G, Taylor D, McLellan J, Smith T, Georgiev I, Tame J, Park SY, Yamazaki M, Gondaira F, Miki M, Katayama K, Murata K* & Kwong P*. (2012) Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle. J Virol 86, 3635-3646. * corresponding authors


Phase plate electron microscopy makes more easy to determine protein structures at nano- and subnanometer resolutions.

Murata K, Liu X, Danev R, Jakana J, Schmid MF, King J, Nagayama K & Chiu W. (2010). Zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and subnanometer resolutions. Structure 18, 903-912.

The structure of alpha1-beta complex in Dihydropyridine Receptor (DHPR: L-type voltage-gated Ca2+ channel) was reconstructed using single particle electron microscopy, which suggests the localization of five subunits in DHPR.

Murata K, Nishimura S, Kuniyasu A, & Nakayama H. (2010). Three-dimensional structure of the alpha1-beta complex in the skeletal muscle dihydropyridine receptor by single-particle electron microscopy. J Electron Microsc (Tokyo) 59, 215-226.

 
   

Electron Microscopes

High voltage electron microscope (Hitachi H-1250M) is used for 3D structural studies of thick biological specimens, like bacteria or cell.
  • Gun: 1250kV LaB6 filament
  • Specimen tilt: +/- 60 degr.
  • Holder: Side-entry Z-rotation/ Liquid N2 Cryo specimen Holders
Phase plate electron microscope (JEOL JEM2200FS) is used to image non-staining biological specimens, like ice-embedded cell, virus particles, or proteins. Zernike phase plate inserted in the objective lens enhances image contrast.
  • Gun: 200kV Thermal FEG
  • Energy filter: Incolumn Omega-type
  • Camera: 4kx4k SlowScan CCD

Serial block-fase scanning electron microscope (SBF-SEM) continuously records the surfance images exposed by triming with the special ultra-microtome. It gives a 3D reconstruction of thick sppecimens, like cell and tissue.

SEM: Zeiss Sigma/VP or Merilin
Microtome: Gatan 3View