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- EMDB-4104: Negative stain reconstruction of the ROD(1-1250):Zwilch:ZW10 -

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Basic information

Entry
Database: EMDB / ID: EMD-4104
TitleNegative stain reconstruction of the ROD(1-1250):Zwilch:ZW10
Map dataNegative stain 3D reconstruction of ROD1-1250:Zwilch:ZW10 complex
Sample
  • Complex: Heterotrimeric complex of Zw10, Zwilch and ROD with a C-terminal truncation of 759 amino acids
    • Protein or peptide: ROD(1-1250)
    • Protein or peptide: Zw10
    • Protein or peptide: Zwilch
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsMosalaganti S
CitationJournal: J Cell Biol / Year: 2017
Title: Structure of the RZZ complex and molecular basis of its interaction with Spindly.
Authors: Shyamal Mosalaganti / Jenny Keller / Anika Altenfeld / Michael Winzker / Pascaline Rombaut / Michael Saur / Arsen Petrovic / Annemarie Wehenkel / Sabine Wohlgemuth / Franziska Müller / ...Authors: Shyamal Mosalaganti / Jenny Keller / Anika Altenfeld / Michael Winzker / Pascaline Rombaut / Michael Saur / Arsen Petrovic / Annemarie Wehenkel / Sabine Wohlgemuth / Franziska Müller / Stefano Maffini / Tanja Bange / Franz Herzog / Herbert Waldmann / Stefan Raunser / Andrea Musacchio /
Abstract: Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona ...Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13-Sec31, and αβ'ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein's cargo at human kinetochores.
History
DepositionAug 22, 2016-
Header (metadata) releaseOct 12, 2016-
Map releaseMar 22, 2017-
UpdateJun 7, 2017-
Current statusJun 7, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.47
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.47
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4104.png

Downloads & links

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Map

FileDownload / File: emd_4104.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain 3D reconstruction of ROD1-1250:Zwilch:ZW10 complex
Voxel sizeX=Y=Z: 4.64 Å
Density
Contour LevelBy AUTHOR: 1.47 / Movie #1: 1.47
Minimum - Maximum-4.2169476 - 17.105537000000002
Average (Standard dev.)0.02075383 (±0.36448944)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 593.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.644.644.64
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z593.920593.920593.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-4.21717.1060.021

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Supplemental data

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Sample components

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Entire : Heterotrimeric complex of Zw10, Zwilch and ROD with a C-terminal ...

EntireName: Heterotrimeric complex of Zw10, Zwilch and ROD with a C-terminal truncation of 759 amino acids
Components
  • Complex: Heterotrimeric complex of Zw10, Zwilch and ROD with a C-terminal truncation of 759 amino acids
    • Protein or peptide: ROD(1-1250)
    • Protein or peptide: Zw10
    • Protein or peptide: Zwilch

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Supramolecule #1: Heterotrimeric complex of Zw10, Zwilch and ROD with a C-terminal ...

SupramoleculeName: Heterotrimeric complex of Zw10, Zwilch and ROD with a C-terminal truncation of 759 amino acids
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 710 KDa

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Macromolecule #1: ROD(1-1250)

MacromoleculeName: ROD(1-1250) / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MWNDIELLTN DDTGSGYLSV GSRKEHGTAL YQVDLLVKIS SEKASLNPKI QACSLSDGFI IVADQSVIL LDSICRSLQL HLVFDTEVDV VGLCQEGKFL LVGERSGNLH LIHVTSKQTL L TNAFVQKA NDENRRTYQN LVIEKDGSNE GTYYMLLLTY SGFFCITNLQ ...String:
MWNDIELLTN DDTGSGYLSV GSRKEHGTAL YQVDLLVKIS SEKASLNPKI QACSLSDGFI IVADQSVIL LDSICRSLQL HLVFDTEVDV VGLCQEGKFL LVGERSGNLH LIHVTSKQTL L TNAFVQKA NDENRRTYQN LVIEKDGSNE GTYYMLLLTY SGFFCITNLQ LLKIQQAIEN VD FSTAKKL QGQIKSSFIS TENYHTLGCL SLVAGDLASE VPVIIGGTGN CAFSKWEPDS SKK GMTVKN LIDAEIIKGA KKFQLIDNLL FVLDTDNVLS LWDIYTLTPV WNWPSLHVEE FLLT TEADS PSSVTWQGIT NLKLIALTAS ANKKMKNLMV YSLPTMEILY SLEVSSVSSL VQTGI STDT IYLLEGVCKN DPKLSEDSVS VLVLRCLTEA LPENRLSRLL HKHRFAEAES FAIQFG LDV ELVYKVKSNH ILEKLALSSV DASEQTEWQQ LVDDAKENLH KIQDDEFVVN YCLKAQW IT YETTQEMLNY AKTRLLKKED KTALIYSDGL KEVLRAHAKL TTFYGAFGPE KFSGSSWI E FLNNEDDLKD IFLQLKEGNL VCAQYLWLRH RANFESRFDV KMLESLLNSM SASVSLQKL CPWFKNDVIP FVRRTVPEGQ IILAKWLEQA ARNLELTDKA NWPENGLQLA EIFFTAEKTD ELGLASSWH WISLKDYQNT EEVCQLRTLV NNLRELITLH RKYNCKLALS DFEKENTTTI V FRMFDKVL APELIPSILE KFIRVYMREH DLQEEELLLL YIEDLLNRCS SKSTSLFETA WE AKAMAVI ACLSDTDLIF DAVLKIMYAA VVPWSAAVEQ LVKQHLEMDH PKVKLLQESY KLM EMKKLL RGYGIREVNL LNKEIMRVVR YILKQDVPSS LEDALKVAQA FMLSDDEIYS LRII DLIDR EQGEDCLLLL KSLPPAEAEK TAERVIIWAR LALQEEPDHS KEGKAWRMSV AKTSV DILK ILCDIQKDNL QKKDECEEML KLFKEVASLQ ENFEVFLSFE DYSNSSLVAD LREQHI KAH EVAQAKHKPG STPEPIAAEV RSPSMESKLH RQALALQMSK QELEAELTLR ALKDGNI KT ALKKCSDLFK YHCNADTGKL LFLTCQKLCQ MLADNVPVTV PVGLNLPSMI HDLASQAA T ICSPDFLLDA LELCKHTLMA VELSRQCQMD DCGILMKASF GTHKDPYEEW SYSDFFSED GIVLESQMVL PVIYELISSL VPLAESKRYP LESTSLPYCS LNEGDGLVLP

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Macromolecule #2: Zw10

MacromoleculeName: Zw10 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
SequenceString: MASFVTEVLA HSGRLEKEDL GTRISRLTRR VEEIKGEVCN MISKKYSEFL PSMQSAQGLI TQVDKLSED IDLLKSRIES EVRRDLHVST GEFTDLKQQL ERDSVVLSLL KQLQEFSTAI E EYNCALTE KKYVTGAQRL EEAQKCLKLL KSRKCFDLKI LKSLSMELTI ...String:
MASFVTEVLA HSGRLEKEDL GTRISRLTRR VEEIKGEVCN MISKKYSEFL PSMQSAQGLI TQVDKLSED IDLLKSRIES EVRRDLHVST GEFTDLKQQL ERDSVVLSLL KQLQEFSTAI E EYNCALTE KKYVTGAQRL EEAQKCLKLL KSRKCFDLKI LKSLSMELTI QKQNILYHLG EE WQKLIVW KFPPSKDTSS LESYLQTELH LYTEQSHKEE KTPMPPISSV LLAFSVLGEL HSK LKSFGQ MLLKYILRPL ASCPSLHAVI ESQPNIVIIR FESIMTNLEY PSPSEVFTKI RLVL EVLQK QLLDLPLDTD LENEKTSTVP LAEMLGDMIW EDLSECLIKN CLVYSIPTNS SKLQQ YEEI IQSTEEFENA LKEMRFLKGD TTDLLKYARN INSHFANKKC QDVIVAARNL MTSEIH NTV KIIPDSKINV PELPTPDEDN KLEVQKVSNT QYHEVMNLEP ENTLDQHSFS LPTCRIS ES VKKLMELAYQ TLLEATTSSD QCAVQLFYSV RNIFHLFHDV VPTYHKENLQ KLPQLAAI H HNNCMYIAHH LLTLGHQFRL RLAPILCDGT ATFVDLVPGF RRLGTECFLA QMRAQKGEL LERLSSARNF SNMDDEENYS AASKAVRQVL HQLKRLGIVW QDVLPVNIYC KAMGTLLNTA ISEVIGKIT ALEDISTEDG DRLYSLCKTV MDEGPQVFAP LSEESKNKKY QEEVPVYVPK W MPFKELMM MLQASLQEIG DRWADGKGPL AAAFSSSEVK ALIRALFQNT ERRAAALAKI K

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Macromolecule #3: Zwilch

MacromoleculeName: Zwilch / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
SequenceString: MWERLNCAAE DFYSRLLQKF NEEKKGIRKD PFLYEADVQV QLISKGQPNP LKNILNENDI VFIVEKVPL EKEETSHIEE LQSEETAISD FSTGENVGPL ALPVGKARQL IGLYTMAHNP N MTHLKINL PVTALPPLWV RCDSSDPEGT CWLGAELITT NNSITGIVLY ...String:
MWERLNCAAE DFYSRLLQKF NEEKKGIRKD PFLYEADVQV QLISKGQPNP LKNILNENDI VFIVEKVPL EKEETSHIEE LQSEETAISD FSTGENVGPL ALPVGKARQL IGLYTMAHNP N MTHLKINL PVTALPPLWV RCDSSDPEGT CWLGAELITT NNSITGIVLY VVSCKADKNY SV NLENLKN LHKKRHHLST VTSKGFAQYE LFKSSALDDT ITASQTAIAL DISWSPVDEI LQI PPLSST ATLNIKVESG EPRGPLNHLY RELKFLLVLA DGLRTGVTEW LEPLEAKSAV ELVQ EFLND LNKLDGFGDS TKKDTEVETL KHDTAAVDRS VKRLFKVRSD LDFAEQLWCK MSSSV ISYQ DLVKCFTLII QSLQRGDIQP WLHSGSNSLL SKLIHQSYHG TMDTVSLSGT IPVQML LEI GLDKLKKDYI SFFIGQELAS LNHLEYFIAP SVDIQEQVYR VQKLHHILEI LVSCMPF IK SQHELLFSLT QICIKYYKQN PLDEQHIFQL PVRPTAVKNL YQSEKPQKWR VEIYSGQK K IKTVWQLSDS SPIDHLNFHK PDFSELTLNG SLEERIFFTN MVTCSQVHFK

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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: 8.5
StainingType: NEGATIVE / Material: Uranyl Formate

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Electron microscopy

MicroscopeJEOL 1200
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 19.0 e/Å2

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 3424

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