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- EMDB-23639: Single-particle reconstruction of a dimer of the Yeast gamma-TuSC -

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Basic information

Entry
Database: EMDB / ID: EMD-23639
TitleSingle-particle reconstruction of a dimer of the Yeast gamma-TuSC
Map dataSingle-particle reconstruction of a dimer of the Yeast gamma-TuSC half map 1
Sample
  • Complex: Single-particle reconstruction of a dimer of the Yeast gamma-TuSC
    • Protein or peptide: Tub4p (gamma-tubulin)
    • Protein or peptide: Spc97p
    • Protein or peptide: Spc98p
Function / homology
Function and homology information


gamma-tubulin complex localization to nuclear side of mitotic spindle pole body / protein localization to mitotic spindle pole body / inner plaque of spindle pole body / microtubule nucleation by spindle pole body / outer plaque of spindle pole body / central plaque of spindle pole body / gamma-tubulin small complex / regulation of microtubule nucleation / karyogamy involved in conjugation with cellular fusion / mitotic spindle pole body ...gamma-tubulin complex localization to nuclear side of mitotic spindle pole body / protein localization to mitotic spindle pole body / inner plaque of spindle pole body / microtubule nucleation by spindle pole body / outer plaque of spindle pole body / central plaque of spindle pole body / gamma-tubulin small complex / regulation of microtubule nucleation / karyogamy involved in conjugation with cellular fusion / mitotic spindle pole body / equatorial microtubule organizing center / mitotic spindle elongation / gamma-tubulin complex / meiotic spindle organization / positive regulation of microtubule nucleation / 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 / protein-containing complex assembly / microtubule / calmodulin binding / protein-containing complex binding / GTP binding / nucleus / cytoplasm
Similarity search - Function
Spindle pole body component 110, C-terminal / Spindle pole body component 110 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 ...Spindle pole body component 110, C-terminal / Spindle pole body component 110 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 110 / Spindle pole body component SPC97 / Tubulin gamma chain / Spindle pole body component SPC98
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.45 Å
AuthorsBrilot AF / Lyon AS / Zelter A / Viswanath S / Maxwell A / MacCoss MJ / Muller EG / Sali A / Davis TN / Agard DA
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-
UpdateMay 19, 2021-
Current statusMay 19, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23639.png

Downloads & links

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Map

FileDownload / File: emd_23639.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle reconstruction of a dimer of the Yeast gamma-TuSC half map 1
Voxel sizeX=Y=Z: 0.6267 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.6259264 - 4.771695
Average (Standard dev.)-0.0010055457 (±0.19277231)
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
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-1.6264.772-0.001

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Supplemental data

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Half map: Single-particle reconstruction of a dimer of the Yeast...

Fileemd_23639_half_map_1.map
AnnotationSingle-particle reconstruction of a dimer of the Yeast gamma-TuSC unsharpened unmasked main map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Single-particle reconstruction of a dimer of the Yeast...

Fileemd_23639_half_map_2.map
AnnotationSingle-particle reconstruction of a dimer of the Yeast gamma-TuSC half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Single-particle reconstruction of a dimer of the Yeast gamma-TuSC

EntireName: Single-particle reconstruction of a dimer of the Yeast gamma-TuSC
Components
  • Complex: Single-particle reconstruction of a dimer of the Yeast gamma-TuSC
    • Protein or peptide: Tub4p (gamma-tubulin)
    • Protein or peptide: Spc97p
    • Protein or peptide: Spc98p

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Supramolecule #1: Single-particle reconstruction of a dimer of the Yeast gamma-TuSC

SupramoleculeName: Single-particle reconstruction of a dimer of the Yeast gamma-TuSC
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Location in cell: Spindle Pole Body
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: SF9
Molecular weightTheoretical: 600 KDa

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Macromolecule #1: Tub4p (gamma-tubulin)

MacromoleculeName: Tub4p (gamma-tubulin) / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGGEIITLQA GQCGNHVGKF LWSQLAKEHA IGTDGLSQLP DSSTERDDDT KPFFRENSRN KFTPRAIMMD SEPSVIADVE NTFRGFFDPR NTWVASDGAS AGNSWANGYD IGTRNQDDIL NKIDKEIDST DNFEGFQLLH SVAGGTGSGL GSNLLEALCD RYPKKILTTY ...String:
MGGEIITLQA GQCGNHVGKF LWSQLAKEHA IGTDGLSQLP DSSTERDDDT KPFFRENSRN KFTPRAIMMD SEPSVIADVE NTFRGFFDPR NTWVASDGAS AGNSWANGYD IGTRNQDDIL NKIDKEIDST DNFEGFQLLH SVAGGTGSGL GSNLLEALCD RYPKKILTTY SVFPARSSEV VVQSYNTILA LRRLIEDSDA TVVFDNASLL NISGKVFRNP NIDLQHTNQL ISTIISSVTN SIRFPSYMYS SMSSIYSTLI PSPELHFLSP SFTPFTSDYI HDDIAHKGHS SYDVMLDLLD PSNSLVSTAM NNPTYFNVYN TIIGNVEPRQ ISRAMTKLQQ RIKFPSWSSS AMHVNIGRRS PYLPLQPNEN EVSGMMLSNM STVVNVFENA CNTFDKVFAK GAFLNNYNVG DLFQSMQNVQ DEFAESREVV QSLMEDYVAA EQDSYLDDVL VDDENMVGEL EEDLDADGDH KLV

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Macromolecule #2: Spc97p

MacromoleculeName: Spc97p / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEIKEVDDRA ELLRYTNNIP LLGKLVNHQP LWSTNPKLKS FSLEKISAPD QRRVQEALVV KDLLNVLIGL EGTYIRYFND YEPSDPETPI EFKIAKKMDP SFKTFSRRIV RYGKQYMILT RAYEKWSDTS FGMVLQRFAY EIRRFLEDVY LKTLVERLER DFNKVPNFSI ...String:
MEIKEVDDRA ELLRYTNNIP LLGKLVNHQP LWSTNPKLKS FSLEKISAPD QRRVQEALVV KDLLNVLIGL EGTYIRYFND YEPSDPETPI EFKIAKKMDP SFKTFSRRIV RYGKQYMILT RAYEKWSDTS FGMVLQRFAY EIRRFLEDVY LKTLVERLER DFNKVPNFSI RELEQIINET EVNKQMELLY NIYEEIFREI EERRTNQSSQ EDFNNFMDSM KNESSLHLRL MVAFDTTVYP VPKGGAILKI FQQKILENLG DRSSVMFLKK LLNNISQDYC TMLYEWLTQG ILNDPYQEFM TYDDLEGKTD NIFDTRDRAW DTQYFIRKDV LLRDCDSEED KNLLFKMLRT GILLKVVRAS LQIPTIPSNS SDITIQEIND FADLMEGSNL ELYVDKCYSR ANEIFLKLFF QGYDLINVLK HLQQIFLGYQ SGHNVLKFLT KNMGELTKHY RNDNNANYDK LLQNFELERQ SENPNNLMRQ LLMIQFDTET LPQVLSHYLQ IYPEVPENNS ANDDSDPLMH ANNFKNMNAI LFDELSKERT GAYHGSNLEL YTPKSAIYHL KFDINIPYPL NIIISRTCMI KYQIILRYQL VLQYHSRLLD ETWMDLNKTP SWKYRGYSHT VKRRIVRATR VLHAKMNHFI KTIMEYFNQN VIDKEVYSLE KCYRNPTLAV AIQNELEGGL TNIMTNRCLS DLIPLQLQIF DIVYKFCKFI KSMRAKLCQL DPVLYEKHKS GMMKTLNEGY RTNNGGQEDV GYQEDAALEL IQKLIEYISN ASSIFRKCLI NFTQELSTEK FDFYDSSSVD AAGIERVLYS IVPPRSASAS SQR

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Macromolecule #3: Spc98p

MacromoleculeName: Spc98p / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MELEPTLFGI IEALAPQLLS QSHLQTFVSD VVNLLRSSTK SATQLGPLID FYKLQSLDSP ETTIMWHKIE KFLDALFGIQ NTDDMVKYLS VFQSLLPSNY RAKIVQKSSG LNMENLANHE HLLSPVRAPS IYTEASFENM DRFSERRSMV SSPNRYVPSS TYSSVTLRQL ...String:
MELEPTLFGI IEALAPQLLS QSHLQTFVSD VVNLLRSSTK SATQLGPLID FYKLQSLDSP ETTIMWHKIE KFLDALFGIQ NTDDMVKYLS VFQSLLPSNY RAKIVQKSSG LNMENLANHE HLLSPVRAPS IYTEASFENM DRFSERRSMV SSPNRYVPSS TYSSVTLRQL SNPYYVNTIP EEDILKYVSY TLLATTSALF PFDHEQIQIP SKIPNFESGL LHLIFEAGLL YQSLGYKVEK FRMLNISPMK KALIIEISEE LQNYTAFVNN LVSSGTVVSL KSLYREIYEN IIRLRIYCRF TEHLEELSGD TFLIELNIFK SHGDLTIRKI ATNLFNSMIS LYYEYLMNWL TKGLLRATYG EFFIAENTDT NGTDDDFIYH IPIEFNQERV PAFIPKELAY KIFMIGKSYI FLEKYCKEVQ WTNEFSKKYH VLYQSNSYRG ISTNFFEIIN DQYSEIVNHT NQILNQKFHY RDVVFALKNI LLMGKSDFMD ALIEKANDIL ATPSDSLPNY KLTRVLQEAV QLSSLRHLMN SPRNSSVING LDARVLDLGH GSVGWDVFTL DYILYPPLSL VLNVNRPFGR KEYLRIFNFL WRFKKNNYFY QKEMLKSNDI IRSFKKIRGY NPLIRDIINK LSRISILRTQ FQQFNSKMES YYLNCIIEEN FKEMTRKLQR TENKSQNQFD LIRLNNGTIE LNGILTPKAE VLTKSSSSKP QKHAIEKTLN IDELESVHNT FLTNILSHKL FATNTSEISV GDYSGQPYPT SLVLLLNSVY EFVKVYCNLN DIGYEIFIKM NLNDHEASNG LLGKFNTNLK EIVSQYKNFK DRLYIFRADL KNDGDEELFL LSKSLR

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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
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: 0.4 µm / Nominal defocus min: 1.0 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 - Sampling interval: 2.5 µm / 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
CTF correctionSoftware - Name: cisTEM (ver. 1.0 beta) / Details: Final reconstruction in cisTEM
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: 2 / Avg.num./class: 593646 / 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: 4.45 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0 Beta) / Number images used: 506014

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Atomic model buiding 1

RefinementOverall B value: 207

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