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1. Í¶ßÉ
¶ÌàÅÍCxÉ§ä³êGlM[IÉñíÉø¦IÈ½ªsíêÄ¢éB¶ÌàÅÌ§ä³ê½½ÉÍC½­Ì@\`¿âj_ÈÇªïµ½lXÈ¶Ì´ªq¡ÌªCSIÈððSÁÄ¢éB±êç¶Ì´ªq¡ÌÍC¡Ìð\¬·é»ê¼êÌ@\`¿é¢Íj_ÅÌ½ÌaÅÍðßÅ«È¢xÈ§änðLµC½ðø¦Ç­sÁÄ¢éB¶ÌàÅÌxÉ§
ä³ê½ø¦IÈ½ÍC´ªq¡ÌàÌ`¿Æ`¿Cé¢Í`¿Æj_ÆÌÔÅÌÝìpª[­ÖíÁÄ¢éB]ÁÄC¡GÈ½nð§äµÄ¢é¶Ì´ªq¡Ìð»ÌÜÜÌ`Å\¢ðÍðs¢Ce@\`¿

Fig. 1

CryoEM reconstruct structure of the rat liver vault. (a) Side
view. (b) Top view.

âj_Ì§Ì\¢zuâÚ×ÈªqÔÝìpÌm©ð¾
é±ÆªC¶ÌàÌgD»³ê½@\ðð·éãÅdv

iªqÊñ10Ì major vault protein (MVPjCªqÊñ19ÅéBX ü»\¢ðÍ@ÍC±êç¶Ì´ªq¡Ì vault poly(ADP­ribose) polymerase (VPARPj
CªqÌÌ§Ì\¢îñð¸xÇ­¾é±ÆªÅ«éÅà­ÍÈ

Êñ29Ì telomerase­associated protein 1 (TEP1jjÆ 1

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éB{eÅÍC¶Ì´ªq»\¢ðÍr[éiFig. 1jBvault ÌCiBL44XUjðp·é±ÆÉæèñÜ­xf[^ÅÍÅåÌ`¿_j_¡ÌÅªûWÅ«½åªq vault Ì»\¢ðÍÉÂ¢ÄÐî

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vault ÍbgÅ­©³êÄÈãCSÛ©çqgÜÅL¢^j¶¨É¨¢Ä©o³ê½B½­Ì¤Ò½¿ª@

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É vault ±qSÌÌ X ü»\¢ðÍÉ`¿âj_ÈÇÌ¶Ì¿Ì»ÍC­ÍÈúËõÉ

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3. ¿²»¨æÑ»»é¢Íê½èC¹ªå«¢Æ«ÉÍS­ÏªÅ«È­ÈéBÁÉCvault ÌlÈåÈ¡ÌÌ»ÍCñÜ­xªñíÉã¢½ßCp¤ÌbgÌÌ©çP£µ½ vault ðp¢ÄClXÈ»

ñÜ_ªÏªÅ«È­ÈèÈèâ·¢B]ÁÄCäXÍÇ

»ððTõµ½B1 xÌ¿²»ÅÍCbgÌ 2 kgÌ»É¨¢ÄàCÜ¸»ðKXLs_Élß©çñ 8 mg ÌxÈ vault ð¾é±ÆªÅ«½B3.5 ÅÄí·ÅÌñÜÀ±ðs¢C»ÌñÜ\âñÜ_Ì`ðªð\Å\¢ðÍª¬÷·éÜÅCêÉ 1`2 ñÌ

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çê½»ÉÂ¢ÄÍCSPring­8 Ì¶Ì´ªq»\¢êéOZ[âG`OR[ClXÈªqÊÌðÍr[C BL44XU Å»ÌñÜ\ðmFµÈª|G`OR[iPEGjÈÇÉÁ¦ÄÞCIçC»»ðÌÅK»ðsÁ½Bªð\ÌñÜ­xCÈÇCñ30íÞÌòðp¢½B»ÌÊCPEG400f[^ª¾çêéÇ¿È»ð¾éÜÅC¿²»yÑªÅKÈRÜÅ»»ðÌÅK»ðJèÔµs¢C6 íÞÌ»ð¾½B

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½BRÜÆµÄgpµ½ PEG400ÌZxÍ34÷iw/vj

3.5 Å ªð\ÈãÌñÜ_ð¦µ½iFig. 2, Fig. 3jB

ÅCÍ»ð34÷ (w/v) PEG400ðÜñ¾ntÉ¼
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xð 5 ª¨«É 1 ÷¸Âã¸³¹Ä»ÖÌ¨IÈ¹ðÉÍ¸ç·û@ðÝ½B»ÌÊCÄ»«æ­¢ªð\ÌñÜ­xf[^ð¾éªÂ\ÉÈÁ½BÜ½C
»ðÅIZxÌRÜÉêÓZ§·é±ÆÅC»ÔÌ¯^«ðßé±ÆÉà¬÷µ½Bvault »ÖÌRÜÌíÞÆZxâû@Ìðªm§µ½±ÆÉæèCªð\ÌñÜ­xf[^ûWÍòôIÉiñ¾B

5. ñÜ­xf[^ûWFig. 2 A crystal of the rat liver vault.

ðÌÅK»Éæè¾çê½Ä»«Ç­ªð\ÜÅ X üðñÜ·é»ÍCóÔQª C2Ciqèª a
702.3 Å, b383.8 Å, c598.5 Å, b124.7ÅÁ½B±Ìæ¤ÈåÈiqðÁ½»ÍCñÜ­xª÷ãÅñÜ_ÌÔuª·¢½ßCñÜ_ðª£µÄªð\ÌñÜ­xf[^ðWßé½ßÉÍCwü«ÌÇ¢PxÌ X
ü¹ªKvÅCåÊÏÌñ³oíðp¢ÄC»ÆoíÊÆÌ£ð·­æÁÄñÜÀ±ðs¤KvªéBÜ½C÷ãÈp¤ÌñÜ_ð¸xÇ­ªè·é½ßÉ
ÍC»ÆoíÊÆÌÔð He KXÉu·µobNO
Ehðº°éKvªéBSÄÌñÜ­xf[^ûWÍ±êçÌðð½µÄ¢é SPring­8 Ì¶Ì´ªq»Fig. 3Diraction image from the vault crystal.

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^r[CÅé BL44XU É¨¢ÄCÊíÌ^pN¿»ÌñÜÀ±ÅÍC1 C[W½èÌU®ð 1ÅbÌIõÅ\ªÅéªCvault ÌlÉåÈ±qÌ

Table 1

Data collection
Resolution (Å)

»ÍñÜ­xªñíÉã¢½ßCªð\Ìf[^ð¾é½ßÉÍU®ð¬³­µC·ÔIõðs¤Kvª
Á½BÀÛCäXªñÜ­xf[^ûWÉp¢½»Åà
1
U® 5 bIõÅÍ 8 Å ªð\öxÌñÜ_µ©¾é±ÆªÅ«È©Á½ªC0.2
U®ÅIõÔð30`60bÉ
Î·±ÆÅÅ3.2 Å ªð\ÌñÜ_ð¾é±ÆªÅ«½
iFig. 3jBµ©µCRÌ±ÆÈªç·ÔIõÉæÁÄ
»ªó¯é X üÉæé¹Íå«­C1 J

½è 5

39

Number of frames
Observed reections

1,361
6,870,966

Independent Reections
Completeness (÷)

1,531,361
92.4

Averaged redundancy

4.5

I/s(I)
RÞ
merge

5.7
0.209

Reection used
Number of protein residues
Þ
R factor (÷)ÞÞ
Rfree factor (÷)Þ½ªÁÄCªð\ÌñÜ­xf[^ZbgÍ·ÔIõÅC¡ÂÌ¯^»ðp¢ÄûW·é±ÆÉµ½BÜ
¸C»Ì¯^«ðmF·é½ßÌt@Xf[^ÆRmsd from ideality
Bond length (Å))
Bond angles (µÄC1
U® 5 bIõÅ 1 ÂÌ»ðgÁÄ 8 Å ªð\öxÌñÜ­xf[^ZbgðûWµ½B·ÔIõÉæ

Ramachandran plot statistics
Residues in most favored region (÷)éf[^ûWÅÍCU®p0.2
Å 1 JÉÂ« 5 ÌñÜ­xf[^ðûWµ½Br[TCYª50 mm~50 mmC

Residues in additionally allowed region (÷)
Residues in generously allowed region (÷)Á½ÌÅC1 ÂÌ»©çñ10xªÌf[^ªûWÅ«½B3.5 Å ªð\

Residues in disallowed region (÷)ÅÌªð\ÌñÜ­xf[^ðWßéÌÉ38ÂÌ»ðgÁ½B
vault »ÌiqÍñíÉå«¢ÌÅCÊíÌ^pN¿»ÌñÜÀ±ÅÍªèµÈ¢50 Å ªð\æèáp¤Éà1000Âß­ÌñÜ_ª

éB±êçápÌæÌñÜ

_ÉÍªqÌO`ð¦·MdÈîñªÜÜêÄ¢éÌÅCáp¤ÌñÜ­xf[^ÌûWÍåÈ¶Ì´ªq¡ÌÌ»\¢ðÍðißéãÅKvsÂÅ

éB

204_
3.5

Number of crystalsRenementöxÌñÜC[Wµ©Wßé±ÆªÅ«È©Á½Bµ»Ì·²ûüÌTCYªñ0.7 mm Å

Intensity data and renement statistics

1,436,394
31,668
0.310
0.330
0.013
1.528
80.0
18.3
1.4
0.3

Averaged B factor
Main chain (Å2)
Side chain (Å2)

112.3

Overall (Å2)

121.4

132.0

ÞRmergeShklSi|Ii(hkl)_
qI(hkl)r|/ShklSiIi(hkl), where Ii(hkl) is
the intensity of reection andqI(hkl)ris the mean intensity of agroup of equivalent reectios.
ÞÞR is conventional crystallographic R factor, S||Fobs|_|Fcalc||/
S|Fobs|. Five percent of the reections that were excluded fromthe renement were used in the Rfree calculaion.

BL44XU ÅÍCr[Xgbpð»©çñ³oíûüÉÚ®µC´ªq¡Ì»Ìáªð\ÌñÜ­xf[^ðûWÅ«éæ¤ÉÈÁÄ¢éBvault Ìáªð\ÌñÜ­xf[^ªèÅÍC¼a0.8 mm Ìr[Xgbpð»©ç200 mm ÌÆ±ëÉÝuµC»Æñ³oíÆÌ£ðªð\ªèÅÍ700 mm ÅsÁÄ¢½Æ
±ëð1200 mm ÉÏ¦C1 C[W½è 1
U®/15bIõÌðÅªèµ½Bt@Xf[^Æ¯^È 1ÂÌ»ðp¢Äáªð\¤200 Å ªð\ÜÅÌñÜ­xf[^ðûW·é±ÆªÅ«½BÅIIÉÍ39ÂÌ¯^»ðp¢Ä200 Å`3.5 Å ªð

Fig. 4

A crystal packing of vault. Packing is shown in the ac plane.

\ÌlCeBuf[^Zbgð¾é±ÆªÅ«½iTable
1jB¯^«ªÈ¢ÈÇÌRÅf[^ûWÉg¦È©Á½»àÜßéÆCêÂÌlCeBuf[^ZbgðûW·6. \¢ðÍ

éÌÉgpµ½»ÌÍSÂÈãÉÌÚéñíÉß©Ân¹ÈìÆÅ

Á½B

6.1\¢ðÍè@

vault Ì\¢ðÍÍCdq°÷¾Ä\¬fðp¢Ä
ªq u·@É æè»i qàÅÌªq ÌÊuð èµiFig. 4jCªqàÌÎÌðgÁ½ñ»wIÎÌiNCS:
Non Crystallographic SymmetryjÉæédq§xÌ½Ï

286  úËõ Nov. 2009 Vol.22 No.6

ðà ¡ ªqÊª1000ÉàyÔ¶ÌàåªqÉÎ·é\¢ðÍ

Fig. 5

Flow chart of phase extension for the vault crystal.
Fig. 7R­factors and correlation coecients between observed andcalculated structure factors from each NCS averaging. The
R­factor (R) in blue and correlation coecients (cc) in red
are plotted against various rotational symmetry values at 10
Å resolution.

i[¬ªiVPARP, TEP1, vRNAjÌÎÌ«ª MVP Éä×Äá¢½ßC8 ññ]ÎÌæèà 3 ññ]ÎÌÅÌ½
Fig. 6

Electron density map by phase extension using 3­fold NCS
axis. (a) Initial electron density map of the half vault at 30 Å
resolution. (b) Obtained electron density map at 4 Å resolu­
tion by phase extension. (c) A cross section on the cap
domain.

o±âèÈæüÏ©çCu¶¨É¨¢ÄC39ÈñÄ¢¤»ÉæÁÄÊüÇ·éû@Åiß½iFig. 5jBvault Ì½ªCdq§xð¶Á­è©Ä¢­±ÆÅsÀª©MÖÆÏ»ÌûªÇ¢ÌÅÍ48{ÅÍÈ­39{År¼[Èª·²ûüÌñ]ÎÌÉÂ¢ÄÍC±qÌ­©ÒÅé L.

ë¤Æðßµ½Bµ©µCLbvhCð\¬·éóÌdq§xÌð¦éÆC»Ì
Á½iFig. 6(cjjB±êÜÅÌè¾éÌ¾ë¤©GvÆ­µYÝàµÏíÁÄ¢­ÌÉ»êÙÇÔÍKvÅÍÈ©Á½B

H. Rome çÌ 8 ññ]ÎÌÆ¢¤àª·NÉnÁÄM¶çêÄ«½10) BÞçÍC±qÌ­©Cdq°÷¾Ï@Éæè|WÅR[gµ½ObhãÅÔÑçÌæ

6.2±qÌñ]ÎÌè@3 ññ]ÎÌÅÌ½Ï»ªdq§xÌ¿ðIÉüã³¤ÉJ¢½óÔÌ vault (Open vault) ðÏ@µ½B»µ¹CLbvÌæÉóÌdq§xª39{ÅÄCÞçªÔÙÌð 8 Æ¦ÄÈCvault Ìñ]ÎçCvault ÌÂñ]ÎÌÍ 3 ñâ39ññ]ÎÌÅ

ÌÍ 8 ññ]ÎÌÅ

é±Æ©
éÆ

éÆ_¶çêÄ«½B2004NÉÍl¦½B±Ì±ÆÌ³«ð§Ø·é½ßÉCäXÍ±q±¤µ½©ðÉîÃ¢ÄNCIdq°÷¾ÅÌ¤Éæ

Ì · ² û ü É 2` 48ñ Ü Å S Ä Ì ñ ] Î Ì ð ¼ è µ ÄèCvault ÌOk

NCS ðp¢½dq§xÌ½Ï»@ÉæèC30 Å ªð\©

Í96ÂÌ MVP Å`¬³êéÆñ³êC»êªèàÆÈç10 Å ªð\ÜÅÌÊg£ðsÁ½B»µÄCÏª³ÁÄ¢½11) BäXàÅÍ»ÌàÉ]ÁÄCªqu·@ê½\¢öqÌå«³ |Fobs | Æeñ]ÎÌÅ½Ïµ½dÅ¾çê½30 Å ªð\Ìdq§xiFig. 6(ajjðC8 ñâq§x©çvZ³ê½\¢öqÌå«³ |Fcalc | ÆÌÔÌ48ññ]ÎÌÅdq§xð½Ï»µÈªç 4 Å ªð\ÜR­factor: RS||Fobs |_|Fcalc ||/S|Fobs | ÆÖW C.C.:ÅÊg£ðsÁ½Bµ©µC¾çê½dq§xÍñíÉ

C.C.S(|Fobs |_|Fobs |)~(|Fcalc |_|Fcalc |)/[S(|Fobs |_êÄ¨èCÆÄàf\zÅ«éàÌÅÍÈ©Á½B|Fobs|)2~(|Fcalc|_|Fcalc|)2]1/2 ðärµ½B³µ¢ñ]Î

äXÍdq§xð½Ï»·éÛÌñ]ÎÌÌð 8 ñ©

ÌÅÍC¼Éä×Ä R­factor Í¬³­ÈèCC.C. Íå«

èC±qÌñ]ÎÌÍ48ññ]ÎÌÅçêÂ¸Â¸çµÄdq§xÌ½Ï»ðÝ½B·éÆC­ÈéÍ¸Å

3 ññ]ÎÌÅÌ½Ï»ÅñíÉ¾ÄÈdq§xð¾é±æéñ]ÎÌÌTõÉæèCvault ª 3, 13, 39ññ]ÎÌéB»ÌÊÍñíÉ¾ÄÅC±Ìè@ÉÆªÅ«½iFig. 6(bjjB4 Å ªð\ÅÌdq§xÅÍCiFig. 7jBðÂ±ÆðÍÁ«èÆØ¾·é±ÆªÅ«½12)

LbvhCÉ a_wbNXð¦··¢óÌdq§xªmFÅ«C{fB[hCÉÍJèÔµ\¢ð¦·dq§xðmF·é±ÆªÅ«½B±Ì 3 Æ¢¤
à48ÌñÅ

é±Æ©çC±qàÉ¶Ý·é}C

6.3±qÌñ]ÎÌðp¢½dq§xÌ½Ï»ÆÊg£±qÌñ]ÎÌª 3, 13, 39ññ]ÎÌðÂÌÅéúËõ Nov. 2009 Vol.22 No.6  287

Fig. 8Comparison between electron density maps averaged by 3­
fold and pseudo­39­fold non­crystallographic rotational
symmetries. The rened MVP model is shown by stickcolored green for carbon atoms, blue for nitrogen atoms andred for oxygen atoms. (a) Electron density map obtained by
the 3­fold NCS averaging is depicted as pink cage. (b) Elec­tron density map obtained by the pseudo­39­fold NCS aver­aging is shown by blue cage.
Fig. 9ÌÅOverall structure of the vault. An MVP monomer is coloredin brown and the others are colored in teal blue.

êÎCÅàÎÌ«Ì¢39ññ]ÎÌÅdq§x

ð½Ï»·éªÅàÇ¢dq§xð¾éû@ÅéªC

äXª¾½»Ì vault ±qÍC»ÌpbLOÉæée¿Å±qªXcñÅ¢½BæÁÄC39ññ]ÎÌ
Å½Ï»·éÆdq§xªêÄµÜ¢CÇ¢dq§xð¾é±ÆªÅ«È©Á½Bµ½ªÁÄCÜ¸ÍÎÌ«ÌÅàá¢ 3 ññ]ÎÌðp¢Ädq§xÌ½Ï»ðs¢Èªç3.5 Å ªð\ÜÅÊg£ðµC¾çê½dq§xðàÆÉf\zðsÁ½B¾ çê ½d q§ xð îÉ f ð \z µ½ Æ± ëC
vault ÌOkð\¬·é MVP ÍSÅ12ÂÌ\¢hCÅ\¬³êÄ¢é±Æª¾ç©ÉÈÁ½BLbvÌæÌ·¢ a_wbNXÍrÅX«ªÏíÁÄ¢½ÌÅ³
ç É 2 Â Ì h  C  É ª ¯ C vault Ì O k ð \ ¬ · éMVP ðÅI13ÂÌ\¢hCÉªÞµ½B¾çê½´
qÀWÉîÃ«ehCÉÂ¢Ä39ÂÌ NCS }gbNXðvZµ½B¾çê½507Âi13~39jÌ NCS }g

Fig. 10

Ribbon drawing of an MVP monomer.

bNXðp¢ÄhC²ÆÉdq§xÌ½Ï»ðµÈªçC6.0 Å ªð\©ç3.5 Å ªð\ÜÅÊg£ðsÁ½B±ÌÛCe NCS }gbNXÍÊg£ÌTCN_ [ ª 25 Å » µ Ä { f B [ ª 175 Å Å²ÆÉ¸§»µ½B±Ì[39ññ]ÎÌðp¢½dq

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Fig. 12Interactions between two half vaults. N­terminal residues ofdomain 1, from Met1 to Glu4, form an intermolecular an­
tiparallel b sheet with those of the 2­fold symmetry­related
molecule.

Fig. 13Structures of MVP shoulder domain and two SPHF
domains. The MVP shoulder domain is structurally similar
to the core domain of stomatin (PhStoCD) and the otillin­2
band­7 domain (FlotBD7). The shoulder domain, PhStoCD
and FlotBD7 overlap within 2.2 Å of root­mean­square devi­
ation of Ca atoms.

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Fig. 11

Hydrophobic interactions between MVP monomers in thecap­helix domain. Most of the hydrophobic residues appear
at the interface between two helices to form hydrophobic in­teractions.

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N. L. Kedersha and L. H. Rome: J. Cell Biol. 103, 699(1986).
A. G. Stephen, S. Raval­Fernandes, T. Huynh, M. Torres,
V. A. Kickhoefer and L. H. Rome: J. Biol. Chem. 276, 23217(2001).
V. A. Kickhoefer, A. C. Siva, N. L. Kedersha, E. M. Inman,
C. Ruland, M. Streuli and L. H. Rome: J. Cell Biol. 146, 917(1999).
V. A. Kickhoefer, A. G. Stephen, L. Harrington, M. O.
Robinson and L. H. Rome: J. Biol. Chem. 274, 32712 (1999).
V. A. Kickhoefer, R. P. Searles, N. L. Kedersha, M. E. Garb­er, D. L. Johnson and L. H. Rome: J. Biol. Chem. 268, 7868
(1993).
G. L. Scheer, P. L. Wijngaard, M. J. Flens, M. A. Izquier­do, M. L. Slovak, H. M. Pinedo, C. J. Meijer, H. C. Clevers
and R. J. Scheper: Nature Med. 1, 578 (1995).
G. L. Scheer, A. B. Schroeijers, M. A. Izquierdo, E. A.
Wiemer and R. J. Scheper: Curr. Opin. Oncol. 12, 550(2000).
S. C. Gopinath, A. Matsugami, M. Katahira and P. K. Ku­
mar: Nucleic Acids Res. 33, 4874 (2005).
H. Tanaka, K. Kato, E. Yamashita, T. Sumizawa, Y. Zhou,
M. Yao, K. Iwasaki, M. Yoshimura and T. Tsukihara:
Science 323, 384 (2009).
N. L. Kedersha, J. E. Heuser, D. C. Chugani and L. H.
Rome: J Cell Biol. 112, 225 (1991).
Y. Mikyas, M. Makabi, S Raval­Fernandes, L. Harrington,
V. A. Kickhoefer, L. H. Rome and P. L. Stewart: J. Mol. Biol.
344, 91 (2004).
K. Kato, H. Tanaka, T. Sumizawa, M. Yoshimura, E.
Yamashita, K. Iwasaki and T. Tsukihara: Acta Crystallogr D
Biol Crystallogr. 64, 525 (2008).
L. Holm and C. Sander: Science 273, 595 (1996).
H. Yokoyama, S. Fujii and I. Matsui: J. Mol. Biol. 376, 868(2008).
N. Tavernarakis, M. Driscoll and N. C. Kyrpides: Trends
Biochem. Sci. 24, 425 (1999).
E. Umlauf, E. Csaszar, M. Moertelmaier, G. J. Schuetz, R.
G. Parton and R. Prohaska: J. Biol. Chem. 279, 23699(2004).
M. P. Kowalski, A. Dubouix­Bourandy, M. Bajmoczi, D. E.
Golan, T. Zaidi, Y. S. Coutinho­Sledge, M. P. Gygi, S. P.
Gygi, E. A. Wiemer and G. B. Pier: Science 317, 130 (2007).

ðà ¡ ªqÊª1000ÉàyÔ¶ÌàåªqÉÎ·é\¢ðÍ ÒÐî Rºh÷åãåw`¿¤É³
E­mail: eikiprotein.osaka­u.ac.jp
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»EBX­ray structure analysis of a 10 MDa biologicalmacromolecule
Eiki YAMASHITA
Koji KATO
Hideaki TANAKA

Institute for Protein Research, Osaka University,
3_2 Yamada­oka, Suita, Osaka 565_0871
Department of Life Science, University of Hyogo,
3_2_1 Koto, Kamighori, Akoh, Hyogo 678_1297
Institute for Protein Research, Osaka University,
3_2 Yamada­oka, Suita, Osaka 565_0871

Abstract Vault, which has a molecular mass of 10 MDa, is the largest cytoplasmic ribonucleoprotein parti­cle. Although vaults are found in numerous eukaryotic species, the cellular function remains unclear. We havedetermined the x­ray structure of rat liver vault at 3.5 Å resolution. The structure consists of 78 identical MVP(Major vault protein) chains with 39­fold dihedral symmetry. Each MVP monomer folds into 12 domains; ninestructural repeat domains, a shoulder domain, a cap­helix domain, and a cap­ring domain. The shoulder
domain is structurally similar to a core domain of stomatin, which is a lipid­raft component in erythrocytes andepithelial cells.

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