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- EMDB-23638: Monomeric single-particle reconstruction of the Yeast gamma-TuSC -

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Basic information

Entry
Database: EMDB / ID: EMD-23638
TitleMonomeric single-particle reconstruction of the Yeast gamma-TuSC
Map dataMonomeric gamma-TuSC single-particle reconstruction main map
Sample
  • Complex: Monomer of the Yeast gamma-TuSC
    • Protein or peptide: Tubulin gamma chain
    • Protein or peptide: Spindle pole body component SPC98
    • Protein or peptide: Spindle pole body component SPC97
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Keywordsmicrotubule nucleation / CELL CYCLE
Function / homology
Function and homology information


inner plaque of spindle pole body / microtubule nucleation by spindle pole body / outer plaque of spindle pole body / gamma-tubulin small complex / regulation of microtubule nucleation / mitotic spindle pole body / equatorial microtubule organizing center / gamma-tubulin complex / meiotic spindle organization / microtubule nucleation ...inner plaque of spindle pole body / microtubule nucleation by spindle pole body / outer plaque of spindle pole body / gamma-tubulin small complex / regulation of microtubule nucleation / mitotic spindle pole body / equatorial microtubule organizing center / gamma-tubulin complex / meiotic spindle organization / microtubule nucleation / positive regulation of cytoplasmic translation / spindle pole body / gamma-tubulin binding / mitotic sister chromatid segregation / spindle assembly / cytoplasmic microtubule organization / mitotic spindle organization / meiotic cell cycle / structural constituent of cytoskeleton / spindle / spindle pole / mitotic cell cycle / microtubule / GTP binding / nucleus / cytoplasm
Similarity search - Function
Gamma tubulin / Gamma tubulin complex component, C-terminal / Gamma-tubulin complex component, C-terminal domain superfamily / Gamma tubulin complex component C-terminal / Gamma-tubulin complex component protein / Gamma tubulin complex component protein, N-terminal / Gamma tubulin complex component N-terminal / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain ...Gamma tubulin / Gamma tubulin complex component, C-terminal / Gamma-tubulin complex component, C-terminal domain superfamily / Gamma tubulin complex component C-terminal / Gamma-tubulin complex component protein / Gamma tubulin complex component protein, N-terminal / Gamma tubulin complex component N-terminal / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Spindle pole body component SPC97 / Tubulin gamma chain / Spindle pole body component SPC98
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBrilot AF / Lyon AS
Funding support United States, 13 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)David Agard United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM031627 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118099 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01 GM105537 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103533 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM083960 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM109824 United States
National Science Foundation (NSF, United States)1144247 United States
National Institutes of Health/Office of the Director1S10OD020054 United States
National Institutes of Health/Office of the Director1S10OD021741 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM124149 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30 GM124169 United States
Department of Energy (DOE, United States)DE-AC02-05CH11231 United States
CitationJournal: Elife / Year: 2021
Title: CM1-driven assembly and activation of yeast γ-tubulin small complex underlies microtubule nucleation.
Authors: Axel F Brilot / Andrew S Lyon / Alex Zelter / Shruthi Viswanath / Alison Maxwell / Michael J MacCoss / Eric G Muller / Andrej Sali / Trisha N Davis / David A Agard /
Abstract: Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In , γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub- ...Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In , γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies, which associate helically to template MT growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC.
History
DepositionMar 17, 2021-
Header (metadata) releaseMay 12, 2021-
Map releaseMay 12, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7m2z
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23638.png

Downloads & links

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Map

FileDownload / File: emd_23638.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMonomeric gamma-TuSC single-particle reconstruction main map
Voxel sizeX=Y=Z: 0.6267 Å
Density
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-2.2619312 - 7.1546426
Average (Standard dev.)-0.005090851 (±0.1951187)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 376.02 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.62670.62670.6267
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z376.020376.020376.020
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-2.2627.155-0.005

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Supplemental data

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Half map: Monomeric gamma-TuSC single-particle reconstruction half map 1

Fileemd_23638_half_map_1.map
AnnotationMonomeric gamma-TuSC single-particle reconstruction half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Monomeric gamma-TuSC single-particle reconstruction half map 2

Fileemd_23638_half_map_2.map
AnnotationMonomeric gamma-TuSC single-particle reconstruction half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Monomer of the Yeast gamma-TuSC

EntireName: Monomer of the Yeast gamma-TuSC
Components
  • Complex: Monomer of the Yeast gamma-TuSC
    • Protein or peptide: Tubulin gamma chain
    • Protein or peptide: Spindle pole body component SPC98
    • Protein or peptide: Spindle pole body component SPC97
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Monomer of the Yeast gamma-TuSC

SupramoleculeName: Monomer of the Yeast gamma-TuSC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Location in cell: Spindle Pole Body
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Tubulin gamma chain

MacromoleculeName: Tubulin gamma chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 52.671188 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGGEIITLQA GQCGNHVGKF LWSQLAKEHA IGTDGLSQLP DSSTERDDDT KPFFRENSRN KFTPRAIMMD SEPSVIADVE NTFRGFFDP RNTWVASDGA SAGNSWANGY DIGTRNQDDI LNKIDKEIDS TDNFEGFQLL HSVAGGTGSG LGSNLLEALC D RYPKKILT ...String:
MGGEIITLQA GQCGNHVGKF LWSQLAKEHA IGTDGLSQLP DSSTERDDDT KPFFRENSRN KFTPRAIMMD SEPSVIADVE NTFRGFFDP RNTWVASDGA SAGNSWANGY DIGTRNQDDI LNKIDKEIDS TDNFEGFQLL HSVAGGTGSG LGSNLLEALC D RYPKKILT TYSVFPARSS EVVVQSYNTI LALRRLIEDS DATVVFDNAS LLNISGKVFR NPNIDLQHTN QLISTIISSV TN SIRFPSY MYSSMSSIYS TLIPSPELHF LSPSFTPFTS DYIHDDIAHK GHSSYDVMLD LLDPSNSLVS TAMNNPTYFN VYN TIIGNV EPRQISRAMT KLQQRIKFPS WSSSAMHVNI GRRSPYLPLQ PNENEVSGMM LSNMSTVVNV FENACNTFDK VFAK GAFLN NYNVGDLFQS MQNVQDEFAE SREVVQSLME DYVAAEQDSY LDDVLVDDEN MVGELEEDLD ADGDHKLV

UniProtKB: Tubulin gamma chain

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Macromolecule #2: Spindle pole body component SPC98

MacromoleculeName: Spindle pole body component SPC98 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 98.336211 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MELEPTLFGI IEALAPQLLS QSHLQTFVSD VVNLLRSSTK SATQLGPLID FYKLQSLDSP ETTIMWHKIE KFLDALFGIQ NTDDMVKYL SVFQSLLPSN YRAKIVQKSS GLNMENLANH EHLLSPVRAP SIYTEASFEN MDRFSERRSM VSSPNRYVPS S TYSSVTLR ...String:
MELEPTLFGI IEALAPQLLS QSHLQTFVSD VVNLLRSSTK SATQLGPLID FYKLQSLDSP ETTIMWHKIE KFLDALFGIQ NTDDMVKYL SVFQSLLPSN YRAKIVQKSS GLNMENLANH EHLLSPVRAP SIYTEASFEN MDRFSERRSM VSSPNRYVPS S TYSSVTLR QLSNPYYVNT IPEEDILKYV SYTLLATTSA LFPFDHEQIQ IPSKIPNFES GLLHLIFEAG LLYQSLGYKV EK FRMLNIS PMKKALIIEI SEELQNYTAF VNNLVSSGTV VSLKSLYREI YENIIRLRIY CRFTEHLEEL SGDTFLIELN IFK SHGDLT IRKIATNLFN SMISLYYEYL MNWLTKGLLR ATYGEFFIAE NTDTNGTDDD FIYHIPIEFN QERVPAFIPK ELAY KIFMI GKSYIFLEKY CKEVQWTNEF SKKYHVLYQS NSYRGISTNF FEIINDQYSE IVNHTNQILN QKFHYRDVVF ALKNI LLMG KSDFMDALIE KANDILATPS DSLPNYKLTR VLQEAVQLSS LRHLMNSPRN SSVINGLDAR VLDLGHGSVG WDVFTL DYI LYPPLSLVLN VNRPFGRKEY LRIFNFLWRF KKNNYFYQKE MLKSNDIIRS FKKIRGYNPL IRDIINKLSR ISILRTQ FQ QFNSKMESYY LNCIIEENFK EMTRKLQRTE NKSQNQFDLI RLNNGTIELN GILTPKAEVL TKSSSSKPQK HAIEKTLN I DELESVHNTF LTNILSHKLF ATNTSEISVG DYSGQPYPTS LVLLLNSVYE FVKVYCNLND IGYEIFIKMN LNDHEASNG LLGKFNTNLK EIVSQYKNFK DRLYIFRADL KNDGDEELFL LSKSLR

UniProtKB: Spindle pole body component SPC98

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Macromolecule #3: Spindle pole body component SPC97

MacromoleculeName: Spindle pole body component SPC97 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 96.940594 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEIKEVDDRA ELLRYTNNIP LLGKLVNHQP LWSTNPKLKS FSLEKISAPD QRRVQEALVV KDLLNVLIGL EGTYIRYFND YEPSDPETP IEFKIAKKMD PSFKTFSRRI VRYGKQYMIL TRAYEKWSDT SFGMVLQRFA YEIRRFLEDV YLKTLVERLE R DFNKVPNF ...String:
MEIKEVDDRA ELLRYTNNIP LLGKLVNHQP LWSTNPKLKS FSLEKISAPD QRRVQEALVV KDLLNVLIGL EGTYIRYFND YEPSDPETP IEFKIAKKMD PSFKTFSRRI VRYGKQYMIL TRAYEKWSDT SFGMVLQRFA YEIRRFLEDV YLKTLVERLE R DFNKVPNF SIRELEQIIN ETEVNKQMEL LYNIYEEIFR EIEERRTNQS SQEDFNNFMD SMKNESSLHL RLMVAFDTTV YP VPKGGAI LKIFQQKILE NLGDRSSVMF LKKLLNNISQ DYCTMLYEWL TQGILNDPYQ EFMTYDDLEG KTDNIFDTRD RAW DTQYFI RKDVLLRDCD SEEDKNLLFK MLRTGILLKV VRASLQIPTI PSNSSDITIQ EINDFADLME GSNLELYVDK CYSR ANEIF LKLFFQGYDL INVLKHLQQI FLGYQSGHNV LKFLTKNMGE LTKHYRNDNN ANYDKLLQNF ELERQSENPN NLMRQ LLMI QFDTETLPQV LSHYLQIYPE VPENNSANDD SDPLMHANNF KNMNAILFDE LSKERTGAYH GSNLELYTPK SAIYHL KFD INIPYPLNII ISRTCMIKYQ IILRYQLVLQ YHSRLLDETW MDLNKTPSWK YRGYSHTVKR RIVRATRVLH AKMNHFI KT IMEYFNQNVI DKEVYSLEKC YRNPTLAVAI QNELEGGLTN IMTNRCLSDL IPLQLQIFDI VYKFCKFIKS MRAKLCQL D PVLYEKHKSG MMKTLNEGYR TNNGGQEDVG YQEDAALELI QKLIEYISNA SSIFRKCLIN FTQELSTEKF DFYDSSSVD AAGIERVLYS IVPPRSASAS SQR

UniProtKB: Spindle pole body component SPC97

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Macromolecule #4: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
40.0 mMHEPES-KOH
100.0 mMPotassium Chloride
2.0 mMMagnesium Chloride
1.0 mMEGTA
1.0 mMGDP
2.5 %v/vGlycerol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: Whatman #1 Filter papers used..
DetailsSample was a mixture of monomers and dimers.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 3.0 µm / Calibrated magnification: 39891 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 31000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 3-100 / Average exposure time: 20.0 sec. / Average electron dose: 76.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3210917 / Details: cisTEM picking
Startup modelType of model: INSILICO MODEL
In silico model: Particle coordinates were semi-automatically picked from filtered and binned images using the e2boxer swarm tool (Tang et al., 2007). Particles were extracted using Relion (Scheres, ...In silico model: Particle coordinates were semi-automatically picked from filtered and binned images using the e2boxer swarm tool (Tang et al., 2007). Particles were extracted using Relion (Scheres, 2012) with a box size of 384 physical pixels resampled to 96 pixels for initial processing. A dataset of ~50,000 particles from 217 micrographs was used to generate 300 2D classes using Relion 1.3. 23 classes were selected and used in the generation of a gamma-TuSC monomer initial model using the e2initialmodel.py function in EMAN2.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1.0 beta)
Final 3D classificationNumber classes: 3 / Avg.num./class: 220924 / Software - Name: cisTEM (ver. 1.0 Beta)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1.0 Beta) / Details: Projection matching in cisTEM
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0 Beta) / Number images used: 398841

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 164
Output model

PDB-7m2z:
Monomeric single-particle reconstruction of the Yeast gamma-TuSC

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