2P540 Single Particle Analysis of Image Data Acquired by Zernike Phase Contrast Transmission Electron Microscope(52. Bio-imaging,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Radostin Danev, Kuniaki Nagayama
2006 Seibutsu Butsuri  
TheBiophysical Society of Japan General IncorporatedAssociation 2P537Mesoscepic model of the transient actin-sliding myosin crossbriclges as eaptured by quick-freeze deep-etch peplica eleetron mlcroscopy OYbshitaka Kimori], YOsuke OguchE Ne:ie Baba2, Eisaku Katayama' 'Inst of Medical Science, The Univ. of Tekyo . 2Dept. ef Electric Engineering. Kogakuin Univ. We have been invcsLigating the trunsieiit struuture of uctin-attached myDsin heads during in vitro sliding. by quick-freeze deep-etch
more » ... ica electron microseepy. We previously obsen'ed that actin-bound myosin heads during sliding have a bent eonfigurutfon to hold actin inside its curvuture, and suggested lhat the realized structure was unlike any crystal structures previeusl}, reperted. Postuluting that the delicate internal pattern revealcd by high centrast rotary-shadowing might refiect the surfaee prefile of the pretein molecules, we cempared the replica images with sllTface-rendered aLomic medels by visual reeognitiun uid found thut acti]attuched structure is similar to that of pPDM-crosslinked myosin in the presence of ADR To morc objcetivcly evaluatc :hc ibasibility ot' our conclusion, we develeped a method to buiid the most likely 3-D model by a combination of protein mculutes after classificaLion and averuging collected rep]icu images yie"'ed from u similar direction, Cry'sta1 strueLure {)r myesin hcad was subdivided inte a niot{]rdomain and a lcvcr-arm and thc imagcs generated from individual parts were compared with the countcrparts in thc replica in]ages, VVb confirrncd that thc orientation of leyer-urm moiety in this unusual configuration is twiste{1 and apart imm the other struetllres. wt also analyzed the images of myosin crossbtidges suppomhg aetin movement in a siniilar manner, mid confirmed our previous conclusion on its close proximity to the novel structure "'c found fer crosslinkcd myesm. 2P5383D TEM of whole cell, Multi Sectien Electron [[bmography C)KazuhiroAoyamai.TornokoTakagi?,IsseiMabuchi2,MasakoOsunti] ,'NI]O/ integratedImagingResearchSuppsrt. Electron tornography based om transmission clectron rnjcroseopy (TIIM> can bc used to provide higiiresolution three-dimensional jnformation about mierostrueturcs. This is an irnportant extcnsion of cenventionaL TEM, which only gives 2D data. Electron tornography has limirations related to rhe mickness af the specimens. When the target materials have a lar.ge volume (i.e. are very thick)such us whote cel]sthe himitation is veTy setious. Thls thickness limitatien can be imprevcd by using ultra high voltas,e TEM. "'here it is pussible to ebserve specimcns up to scvcraL micremctcrs thick, Howevcr, thc cxtrcmely high cost makes this appreaeh impructicul, und the thickness limitution still remains. By contrast the method of 3D structurat reconstruction frorn a seties of images, taken from ultra thin sections, has been used for 3D strucLu:al analysis aL the cell le}'el sincc historical days. By applying this incthod to clcctron tomography. thc tiniitation of thiekness can be cempletely everceme eyen with a standard electron micToscepe. Fu]ly autemuted tomography systems including reconstruction softw"Te is now uvailable. It is thus possible to do mere complicated 3D rcconsrmctions, using a 1arge number of elecrron tumography data set, In this study. huge 3D velume was revealed frorn S sets of 3D rcconstrueted data from a sefics ef sumip]e slices. We calL this method 'iMultj Section ElectTon TomographY'. 2P539Development of GUI-based too]s for three dimensional electron tomegraphy Dynainies Prqject, RIKEN Harima Institutc, 3Institute for Protein Rcsearch. Osaka Univcrsity Threc dimensiunal recunstruction from tilt-series micrographs ebtained by electren mierescopy is eallcd elcctron cemputcd tomography <CT). -ris niethed has advantages. e.g, three dimensional reconstruction cloes not require deterniination of panicle retational anglcs andior any symmctry. To rcconstruct three dimensiunal structure of a whule visual field by the CT, some udeguute applications have bccn prcviously rcpvrted, No utici'ul applicatiuns, helvevei, "'cre propesed tbr CT of individual biemolccules and se it is neecssa:}, to combinc two oi' niore different applications so far. Hence, we need to learn rwo or more softvs'are and convert munv fite formats into each other. In addilion. there were serinus problenls that artificial mistakes weTe geneTated by the manuul operution during time-consuming and laborious iterative proccssing, To solvc thcsc problems, we deveioped u titt-series hnage vieiver which call easily discover mistakes in particle selecLions and an integTated too] that uutomates processes for three dimeiisional reconstruction of sing]e moLecules as possible. All of irnage prncesses were inte.ffrated into the latter tool by using UNIX 'makei. Moreover, we huve deveioped a graphical user interface (GUI) tool as well. As a rcsulL our dcyclepcd systcins ghortened heuTs [}f work and redueed urtifieiat mistalces. We hereby could build up the newty deve]opecl integratiun environment t'or electTon temograpby ef s;n.vle molccules, 2P54eSingle Particle Analysis of lmnge Data Acquired by Zernike Phase Contrast Tkransmissien Electron Microscope ORadostinDancy.KuniakiNagayama Okazak Institute fnr Integrative Bioscience, National Institutes nf Natural Sciences (NINS) Wle discuss the application of Zernike Phase Contrast Transmission Electron Microscope (ZPC-TEM) to acquisi:ion of iniagc data t'er singlc pcu'ticlc analysis ot' biolegical sumples.The ZI]C-TEM utilizes a Zernike phase p]ate [1-3] which consists of a thin film with a smalr hule in Lhe center. It is pusi[ioned at the backfocal plane of the objectivc lcns. This phasc platc introduccs half pi phasc shift to thc scattered clectrons ]caving thc ccntral bcam of unseattcred elcctrons intact. Thc iesulting ima.oes exhibit rnuch higher overull contrast due to the presence of lew sputiul i're{tuellcies which ure strongay attenuuted in images ucquired using u conventional TEM, The ZPC-TEM has overal1 "fiati' ftequency response compared to the "'band-pass'i filtcr eharactcristics of the convcntional defbcus phase contrast TEM.Data and resu]ts nf sing]t particlc analysis usin.g ZPC-TEM images are pTesented. Theoretical and practical aspects in the application of ZPC-TEM are
doi:10.2142/biophys.46.s430_4 fatcat:iq5of7jmbnfnddzyafietvhrqu