2P027 The crystal structure analysis of Aquifex aeolicus methionl-tRNA synethetase(29. Protein structure and dynamics (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)
TheBiophysical Society of Japan General IncorporatedAssociation 2P025Development of Online UV-Visible MicrospectTophotometer for Protein Crystaltography at BL38Bl of SPring-8 ONobataka Shimizui, Ko"ichl Tbmita2, Masao Ogalti3, Kazuya Hasegawai, MasakiYtmamotoiA iJASRVSPring-8,2PHOTONDcsignCo"]EI'LInc"'RIKENSPring-8Center The high intensity X-ray ef sy"ehretron radiation gives the radiatien damagc to a protein crysta1. It is di fficult to evaluate the damuge througb structura! analysis, and is
... eessary to obtain the complementary information of the damage against the Xruy difftaction experiment. UV-visible spectroscopy is onc of good techniques for ubtaining the infbrmutien of umino acids in the protein. Especiatly, Trp and Tyr havc absorptien around 2gO nm unlike other amino acids. Moreever, since preteins which bind with metal atoms and organic smaN molccules have absorptiou in the visible region, thc changc of specifie pasition in the pretein might be clarified. We developed online UVLVis. microspectrophotorneter at BL3SBI ot' SPring-g to analyrc thc staLe ut' protein in the crystal to which X-ray was irradiated. This spectrephetometer cmployed a diffcrcnce dispersive double monochrometer mounted by Czerny-Thrner type in order to acquire high brightness in UV region and reject stray light (PDPIr]320, PHOTON design). Two typcs of ditl'raction gratings (53006BKOI-150R and 53006BKel-2SOR, Newport) wcTc installed into the monochrometer, Mercury-Xcon lamp (L2423, Hainamatsu Photonics) and the phetomultipller CR374. Hamamatsu Photonics) arc selccted as a light source and a detector, respectively. In the prelifiinary test using solution samples and ND filters. the absorption spectra were ab[e to be rneasllred up to fi-3.e OD from 250 tD 7ee nm. We will introduce the feature of this spectrophotorneter and the rneasurernent results of crystals on the poster. There are 9 beamlines for protcin crysmi,lography at SPring-S as ei' Mtty 2006. Two beamlines, BL3gB T (StructLra1 Biology fll) and BL4 [XU (Structural Bielegy I), out of them are dedicated for public uses. Ihe BI.38B1 is designcd based on u standurd SPring-8 bellding magnet beamline. consisting ef a Si(111} doublc crystal menochrometer and a lm-long Rlt-coated cy]indrical bend inhTor. and used for routine protein crystallography, including thc 'iMail-in data collectien system ". The instalLation of sumple changer rebetics, eontrol and database system have already finjshed. Now we are aving final check of the entire system. The BL41XU is an undutator beamline using the SPrjng-8 standard in-vacuum undulator as the ]ight source. The undulator beum is monochromatized by rotatedinclined Si(111) double crysta] monochrometer, and ftrcused by KB mi.rrprs, UrilLzing this high bti1liant X-ray beam, we are foeusing on a measuremcnt in uttrahigh resohition and a data collection from rnicro-erystals, Thc 1arge area detecters and very short wavelength X-ray from undulator 3rd harmonjcs cnablcs to collect difimction $pc}ts over O.5 A reso]utioll. We have succeeded to collect a dataset of Endopolygalacturomase I at O.6S A resolution (R",. = 1O.S%). To collect eencient qualjty data ftom micre-crystals, it is necessary to use ntiero-beam to teciuce the bttckground noise. Present]y, the size of 25 um beam with ..1Oii photonslsec was achieyed, and we ceuld cellect a dataset frem lysezyme crystal sized .-20 -m up to o1.9 A resolution B..,. = 6%) 2P027The crysta1 structure analysis ofA4zadex aeelicus methionl-tRNA synethetase OKotaroNakanishi],YasuhjroKomotoi,Shuyallakai',OsamuNurekii2'i J[)ept. of Biologjcal Informatien, Tokyo lnstitute et Tcchnology, !PRESIO, JST, SRIKENGenomicSciencesCenter Aminoacyl-tRNAsynthetase (aaRS) charges un arnino acid to the 3' terminal of the cognate tRNA, which p]ays thc esscntial rele in the transltttion ffom codon to amintmeid. NVle reperted the first crystal strueture ofAquCfex aeolicus methionyl-tRNA synthetase (MetRS)-tRNA"TeZ.-methionyl-AMP analog tertiary cemptex in last BSJ conference, The structure revealed how MetRS recognitied anLicc}don bases, C34, A3fi and U36, known as important identities of tRNA"i" for MetRS, However, the electron density maps of either the CCA terminal aL thc 3' end of tRNA""t or the eonnective polypepLide 1 <CP]) demain of MetRS were disordered, In addition. theA73 called the discrirninator, whieh is t]ie -ucleetide preceding the CCA tcrminal and has been theught to be cnicial base for aminoacylation by MetRS, had no interaction with residue ot' MetiRS, Thus, this structure might indicate the pre-umineacylation form of MetRS-tRNAN'iet complex jllst after binding and before preceding the penetration of the 3' emd of tRNA inte the active site of MetRS. Tb elucidate the mechanism that the discfuninatoT and the CCA te]Juina] were recognized by MctRS, we tried to crystallize, and then determine the new crystal structLire of A, aeoticus MctRS-tRNAb]et binary complex that provided more distillct elecnic density map ofCPI domain. Now, the model is undcrrcfincment, 2PO28 Crysatal Structural analysis of HindllI restriction endonuclease in o complex with cognate DNA at 20 A resolution OChika Satoi, Nebuhistt WILtanabe:'2, Yozo TakasakiS, [sao Tanakai'L' iDiv, of Biol, Sci., Grad, Sch. of Sci" Hokliaido Univ., 2Fac. of Adv. Life Sci., Hokhaido Uniy" iDep, ef Biomel, Sci" Fae of Med" Saga Univ, Restriction endonucleases, which arc gomponcnts of rcstriction modification systems thaL protect bacteria and arehaea uguinst invading forcign DNA. arc divided into 4 grollp$ by their subunit cumpesition and eufactur requirement. Type 1I restriction endonucleuses are usually homodimcric er homotctrameric enzymes, They llsllally recognize 4 or 8 palindromic sequences and cleave DNA in both strunds. To cleftve DNA, lhey require Mg" as a cofactor and do not usc MP or GTR }tLndln, whose subunit consists of 300 atninu acids with a molecular rnttss of 35 kDa, is one of the type Il restriction cndonuclcases and recegnizes the palindromichexanucleotidesequenceAIAGCTT, Up te now. out of over IOOe kinds of type II restriction eiidnucleases, threedimensiontd structures of less thErm 2e of them have been determined. It was rcvcalcd that they had a common sequence motif that needs for their caialytic activity. However. the recognitien mechanism of cognatc sequcncc, thc rrrechanism of cleaving DNA and tjie number efMg]' thut need to cleave DNA ef Hindlll werc sdilunknown, In order to elucidate its DNA recognition mechanism, we have crygtaNi7ed Hind][lcogmare DNA eomplex in Mg7' free form, The space group of the crystal is P2" and the unit-cell parameters are a = 83.9, b = 130.9, c = 93,6 A, and P = 110. 2". Thc crysta1 structure was solved at 2.9 A resolution with the SAD methed using iodinftted tyrosine as anemalous scatterers, and a $tucture refillement is ill prDgress with 2.o A native dataset.