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- EMDB-21912: Mdn1-DeltaC plus MIDAS -

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Basic information

Entry
Database: EMDB / ID: EMD-21912
TitleMdn1-DeltaC plus MIDAS
Map data
Sample
  • Complex: Mdn1(1-3911) plus SNAP-MIDAS
    • Complex: Mdn1(1-3911)
      • Protein or peptide: Mdn1(1-3911)
    • Complex: SNAP-tagged MIDAS domain of Mdn1
      • Protein or peptide: SNAP-tagged MIDAS domain of Mdn1
Biological speciesSchizosaccharomyces pombe (fission yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 25.55 Å
AuthorsMickolajczyk KJ / Niu Y
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM98579 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM109824 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM130234-01 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)K00CA223018 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103314 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1.
Authors: Keith J Mickolajczyk / Paul Dominic B Olinares / Yiming Niu / Nan Chen / Sara E Warrington / Yusuke Sasaki / Thomas Walz / Brian T Chait / Tarun M Kapoor /
Abstract: Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an ...Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.
History
DepositionMay 4, 2020-
Header (metadata) releaseJul 22, 2020-
Map releaseJul 22, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0481
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0481
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21912.png

Downloads & links

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Map

FileDownload / File: emd_21912.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.71 Å/pix.
x 200 pix.
= 542. Å		2.71 Å/pix.
x 200 pix.
= 542. Å		2.71 Å/pix.
x 200 pix.
= 542. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.71 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0481 / Movie #1: 0.0481
Minimum - Maximum-0.05648675 - 0.1354544
Average (Standard dev.)-0.00050864305 (±0.005570627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 542.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.712.712.71
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z542.000542.000542.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0560.135-0.001

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Supplemental data

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Sample components

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Entire : Mdn1(1-3911) plus SNAP-MIDAS

EntireName: Mdn1(1-3911) plus SNAP-MIDAS
Components
  • Complex: Mdn1(1-3911) plus SNAP-MIDAS
    • Complex: Mdn1(1-3911)
      • Protein or peptide: Mdn1(1-3911)
    • Complex: SNAP-tagged MIDAS domain of Mdn1
      • Protein or peptide: SNAP-tagged MIDAS domain of Mdn1

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Supramolecule #1: Mdn1(1-3911) plus SNAP-MIDAS

SupramoleculeName: Mdn1(1-3911) plus SNAP-MIDAS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: MAP of Mdn1(1-3911) with MIDAS domain bound to the AAA ring
Molecular weightExperimental: 453 KDa

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Supramolecule #2: Mdn1(1-3911)

SupramoleculeName: Mdn1(1-3911) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Supramolecule #3: SNAP-tagged MIDAS domain of Mdn1

SupramoleculeName: SNAP-tagged MIDAS domain of Mdn1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Mdn1(1-3911)

MacromoleculeName: Mdn1(1-3911) / type: protein_or_peptide / ID: 1 / Details: Rbin-1 / Enantiomer: LEVO
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMGIRN SKAYVDMDVL IEWVAIYPQI YDILEHINYV PSNTLQRLRL HQPWSKIDYD VWFLYASDEI RETCKVKYYG ETKTYGEVFV LENERISQLH RLFVSWTVSE RAEHLKNLLF DAGLSNLPLV ELGGNVFFNS HVPLPCSLVL ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMGIRN SKAYVDMDVL IEWVAIYPQI YDILEHINYV PSNTLQRLRL HQPWSKIDYD VWFLYASDEI RETCKVKYYG ETKTYGEVFV LENERISQLH RLFVSWTVSE RAEHLKNLLF DAGLSNLPLV ELGGNVFFNS HVPLPCSLVL TKSTQENLNR ITPYLVQKRP ILLAGPEGIG KKFLITQIAA KLGQQIIRIH LSDSTDPKML IGTYTSPKPG EFEWQPGVLT QAVITGKWIL FTNIEHAPSE VLSVLLPLLE KRQLVIPSRG ETIYAKGSFQ MFATSSMKTK ILGQRLWQIL DLTYQPDECV EVVSTLYPVL SIICPTLYSV YKDIFDLFSQ RSFLATSKIY RRLCLRDFYK FIKRVAFLYH KFMIPSDHVV ISQELQDAVF KEAIDMFGAF IPSRDGFDLV VRNVAIELNI PPEKALQLRY SIPVFQNLEH NINIGRCSLK KLSTIRSCST NSYAFTSSSL GLLEQLAAGV QTNEPLLLVG ETGTGKTTTI QLLAGLLGQK VTVINMSQQT ESSDMLGGYK PINASTLGLP LHERFIDIFE QTFSSKKNAK FISMASTSAR RFRWKTCLKI WKEACKLSKT VLDGQQPLPN PQKRQKRLSN QVELRNQWAK FEKEVEDFEK VLTGGSNGFM FSFVEGALVK AVRSGHWVLL DEINLASLET LEPIGQLLSS YESGILLSER GDITPITPHK NFRLFGCMNP STDVGKRELE PSFRSRFTEI YVHSPDQNLD DLLSIIQKYI GSLCIGNEHV IREVAELYQV AKSLSLDGSL VDGAGQRPHY TVRTLSRTLS YVTEIAPIYG LRRSLYEGFC MSFLTLLDHT SESLLYNHVV RFTLGELNRD QQNAILKQIP KVPDHSSYIA FCHYWLRRGS FPVEEQEHYI ITPFVQKNLL NIARACSTRM FPILIQGPTS SGKTSMIEYV AKKTGHKFVR INNHEHTDLQ EYIGTYVTDD NGSLSFREGV LVEALRNGYW IVLDELNLAP TDVLEALNRL LDDNRELFIP ETQVLVKPHP EFMLFATQNP PGVYAGRKHL SRAFRNRFLE IHFDDIPENE LETILHKRCK IAPSYAAKIV QVFRELSLRR QTTRIFEQKN SFATLRDLFR WAFREAVGYQ QLAENGYMLL AERARDQKDK LAVQEVIEKV MKVKIDTDGI YNLDSMEIFQ DMSLKEGPLS KVVWTRPMIR LFCLVWRCLL AKEPVLLVGD TGCGKTTVCQ ILAECLHKEL HIINAHQDTE NGDIIGAQRP VRNRSAVNYS LHSQLCEKFN VQESLDSIDD LIEKFEKLSS SEKNDNLSNL IERQIIKYRS LFEWHDGALV TAMKQGDFFL LDEISLADDS VLERLNSVLE LSRTLTLVEH SNAAVSLTAK DGFAFFATMN PGGDYGKKEL SPALRNRFTE IWVPPMVDTE DILKIVEGKL HNNKIELARP LVEYAKWHAN EYLYTDVISI RDVLSAVEFI NACEILDLNL VLFNAVSMVF IDALGSFTTF SLSNNLASLH AERQRCFAKL NELAGSNIMA SKSADISIKF SDSSFFIGDF GIPLGDSVES DSTYSLHTDT TLMNASKVLR ALQVLKPILL EGSPGVGKTS LITALARETG HQLVRINLSD QTDLMDLFGS DVPVEGGEGG QFAWRDAPFL AAMRNGHWVL LDELNLASQS VLEGLNACLD HRNEAYIPEL DKVFKAHPNF RVFAAQNPQH QGGGRKGLPR SFINRFSVVY VEALKEKDMI EIAACNYHQV NEDWRLKIIK FMFRLQDNIE KDISFGSFGS PWEFNLRDTL RWLQLLNDAP KYTCVSPADY LEVMVLHRMR TVEDRVRTCE LFKEVFDIDY EPRTIGFSLS SQCFKVGHSL LVRDVERQKT LLDSQNILQS QLPVLESVIT CINKKWPCIL VGDTATGKTC ILRLLAAIAG AKIKEMAVNS DTDTMDLIGE YEQIDISRKA SELFTDLSQQ LLNIVIKYRN FDNIFRETSL YTLTTTSFKT HSQAFTLLQK VVDQLDQLKI HETLVHSLGD IHEKARKLLA EFSASPAGRF EWFDGYLLKA VEEGHWFVLD NANLCSPAVL DRLNSLLEHK GVLIVNEKTT EDGHPKTIKP HPNFRLFLTV NPVYGELSRA MRNRGVEIFL LKEALTEIDK KQMSLLEPAP ISSAVDTLAS NISYIKYVFE TMGKIEIDGN YMYIAHAIIL ALFSPRQLKL LRKVLLTNPQ FSLSIKADAE LLLTLKNLVQ KIYCADYFNH MDLKASRFMD IYEYPVQLRE VVGLIQTIND FQSVILTSHL ELPETYASGL LFVSAHEILD LTEEVNRLAV STSNSTYLLK SASAVYHNVS SFKGSTPSLW NLLNQFSKFL IEIASANSNI VYKLSYDVIR HFLKLVVLWK NIYVWTNVPD CDISRFYCYT KMLGEWMFTL TEKTKLLESF LPKDSLEKFS ELQNLSTGLH MQAIWDKWHA FVPRTYDQWS LWNTVDKLLT QYVNANIPSI SMETTACEVV GTSLSLLNKV LVENEVGDIY SYLKILGKGV NELKSSKQVI LPENLVNLFN CLASLDLLHI FIKYTTSSFF LTDDFVRFIR VCFHSRISGN LLTLLHGISF DSTKAVAPVL TYFDFCSLTT GNILGRIALA FTSIDENANL ESANIFEHAR LALLQHFMDH SSLLAEDSST KMNLILLQRY AVIISIFLDQ GKCEKANDLI TKLSLPYEEL AENFVSILEA CKAFLVANSE FISYTYTERF IHSLRFLKDS WLSSNQQKML KNQGMAYIYF ASGMLLVYVP DKPFDPALLP LLTVESLRHY LESLYKESQI LEIAESLNSG KVNSVMRRLV STEISNTPNI DSSFSTVYRS LNESIVPLYS ELEFFMKSVV LNQYIFELAM RLSKESNIAV VEEAKSFVTK WKAYIERIRE AYPQFVDVYE LILSFISFMI YGIELLMFEA KRRLDERSQI LSTLILTLVD PSSFARSLSF DDVSNLIEQI KVLDLNDSIR FEIYLFLASR LCSEKQHSSD THSLANSFVL LANEFYIHNA KIKQKELEEI EEKNRLYRQR EFNFDKNDYL KVFINYDDEV EPEVEPEVVI ERKRFLQLQF AFWSLYNEIY SEKMNVIPLE QLMNTGSYLA KKIKVKNPDM IASSGFDIVS VVLMMGVKST NERQYWTPPV YNFYSDPNPS KAIEVRDLIK IVESRAISLI KNWPENFVLR GLKDAIDAIL NLSPFSPIAE YLSKLERVFH LLSEWEKLAS REYSLANEMD LIKKKIIDWR KFELSNWNNL LKLEEYKLSE RVYPRLYSIL QFIILKPFFE NSKFTKQNLC ESASIIVQFI TDLTVGEFQL CLKCLLSFSQ HAASLRICHG IDAMLLNIYH YFEQFLSKVS EAIHTQKQSL ENSIKERILL MSWKDTNVYA LKESAKKSHA ELFKVLHRYR EVLRQPVSSY LSQKHDWDSL LDTENNSAMW VAKKVNLSPS YIEKMDTEIM KLVPVRFSNT PTTLRLMWTL FANVEKPGST FTNMVSNLIT DARELMKLTP ETINDDNLSE IKHLKSRKHL LLTETFKTLK AFGLQYRVKA GIEENLSNLR NLLAVIPTFP VTSLSIEKVD RSLMKSLDFI PKFQTLAGHQ HNDLSVPEVQ KGVGLFNSML SLQLGERAQL VEFTNELLAL KNVYSEVGVN GSPLESFNNS SFNEVSSLGY DHDFENRAQA VSMLCQIYAI VIQKHSSISP TASFQSIGHE LSRFADLLSN KLFPSSIPLY ASADKVSSIR DQQKGINDLI EYCRKKRTEL PELSYCFKHL VSLQSLKSIS RTQVDLTNDE FLNLMNFVLN LFDSLLSSIE TATKNMRTFK ELAETSSFIE MSSCFSKVLR AFNLKFQSMK LSSLKEKLRS SSVDKMSCQL LMLFLPVCEQ FINLAESVLD YFINVHNSNL DSLSKISTLF FMVANNGFCS PDLPQEGKSN SGELESG

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Macromolecule #2: SNAP-tagged MIDAS domain of Mdn1

MacromoleculeName: SNAP-tagged MIDAS domain of Mdn1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GMDKDCEMKR TTLDSPLGKL ELSGCEQGLH EIKLLGKGTS AADAVEVPAP AAVLGGPEPL MQATAWLNAY FHQPEAIEEF PVPALHHPVF QQESFTRQVL WKLLKVVKFG EVISYQQLAA LAGNPAATAA VKTALSGNPV PILIPCHRVV SSSGAVGGYE GGLAVKEWLL ...String:
GMDKDCEMKR TTLDSPLGKL ELSGCEQGLH EIKLLGKGTS AADAVEVPAP AAVLGGPEPL MQATAWLNAY FHQPEAIEEF PVPALHHPVF QQESFTRQVL WKLLKVVKFG EVISYQQLAA LAGNPAATAA VKTALSGNPV PILIPCHRVV SSSGAVGGYE GGLAVKEWLL AHEGHRLGKP GLGKLAAAGE DTLPTEFGSI NQSEKVFELS EDEDIEDELP DYNVKITNLP AAMPIDEARD LWNKHEDSTK QLSIELCEQL RLILEPTLAT KMQGDFRTGK RLNMKRIIPY IASQFKKDKI WMRRVKPSKR TYQVMISIDD SKSMSESGST VLALETLALV TKALSLLEVG QIAVMKFGEQ PELLHPFDKQ FSSESGVQMF SHFTFEQSNT NVLALADASM KCFNYANTAS HHRSNSDIRQ LEIIISDGIC EDHDSIRKLL RRAQEEKVMI VFVILDNVNT QKKSSILDIK KVYYDTKEDG TMDLKIQPYI DEFAFDYYLV VRNIEELPQL LSSALRQWFQ QMSNT

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
StainingType: NEGATIVE / Material: uranyl formate

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Electron microscopy

MicroscopeFEI/PHILIPS CM10
Image recordingFilm or detector model: OTHER / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

DetailsXR16L-ActiveVu
Particle selectionNumber selected: 13000
Final reconstructionResolution.type: BY AUTHOR / Resolution: 25.55 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 5494
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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