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- EMDB-10744: Microtubule Nucleation by Single Human gamma-TuRC in a Partly Ope... -

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Basic information

Entry
Database: EMDB / ID: EMD-10744
TitleMicrotubule Nucleation by Single Human gamma-TuRC in a Partly Open Asymmetric Conformation
Map dataCryo-EM structure of human gamma-TuRC
Sample
  • Complex: gamma-tubulin ring complex
    • Protein or peptide: gamma-tubulin ring complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsLocke J / Costa A
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)820102 United Kingdom
CitationJournal: Dev Cell / Year: 2020
Title: Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure.
Authors: Tanja Consolati / Julia Locke / Johanna Roostalu / Zhuo Angel Chen / Julian Gannon / Jayant Asthana / Wei Ming Lim / Fabrizio Martino / Milos A Cvetkovic / Juri Rappsilber / Alessandro Costa / Thomas Surrey /
Abstract: The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in ...The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.
History
DepositionMar 11, 2020-
Header (metadata) releaseJun 3, 2020-
Map releaseJun 3, 2020-
UpdateJun 17, 2020-
Current statusJun 17, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10744.png

Downloads & links

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Map

FileDownload / File: emd_10744.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of human gamma-TuRC
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.005
Minimum - Maximum-0.049079012 - 0.062492684
Average (Standard dev.)0.00005164352 (±0.00091257173)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 552.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z552.960552.960552.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0490.0620.000

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Supplemental data

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Sample components

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Entire : gamma-tubulin ring complex

EntireName: gamma-tubulin ring complex
Components
  • Complex: gamma-tubulin ring complex
    • Protein or peptide: gamma-tubulin ring complex

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Supramolecule #1: gamma-tubulin ring complex

SupramoleculeName: gamma-tubulin ring complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HeLa-Kyoto cells
Molecular weightTheoretical: 2.2 MDa

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Macromolecule #1: gamma-tubulin ring complex

MacromoleculeName: gamma-tubulin ring complex / type: protein_or_peptide / ID: 1
Details: Complex consists of 5x native GCP2/GCP with mBFP tag, 5x GCP3, 2x GCP4, 1x GCP5, 1x GCP6 and other factors
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSEFRIHHDV NELLSLLRVH GGDGAEVYID LLQKNRTPYV TTTVSAHSAK VKIAEFSRTP EDFLKKYDEL KSKNTRNLDP LVYLLSKLTE DKETLQYLQQ NAKERAELAA AAVGSSTTSI NVPAAASKIS MQELEELRKQ LGSVATGSTL QQSLELKRKM LRDKQNKKNS ...String:
MSEFRIHHDV NELLSLLRVH GGDGAEVYID LLQKNRTPYV TTTVSAHSAK VKIAEFSRTP EDFLKKYDEL KSKNTRNLDP LVYLLSKLTE DKETLQYLQQ NAKERAELAA AAVGSSTTSI NVPAAASKIS MQELEELRKQ LGSVATGSTL QQSLELKRKM LRDKQNKKNS GQHLPIFPAW VYERPALIGD FLIGAGISTD TALPIGTLPL ASQESAVVED LLYVLVGVDG RYVSAQPLAG RQSRTFLVDP NLDLSIRELV HRILPVAASY SAVTRFIEEK SSFEYGQVNH ALAAAMRTLV KEHLILVSQL EQLHRQGLLS LQKLWFYIQP AMRTMDILAS LATSVDKGEC LGGSTLSLLH DRSFSYTGDS QAQELCLYLT KAASAPYFEV LEKWIYRGII HDPYSEFMVE EHELRKERIQ EDYNDKYWDQ RYTIVQQQIP SFLQKMADKI LSTGKYLNVV RECGHDVTCP VAKEIIYTLK ERAYVEQIEK AFNYASKVLL DFLMEEKELV AHLRSIKRYF LMDQGDFFVH FMDLAEEELR KPVEDITPPR LEALLELALR MSTANTDPFK DDLKIDLMPH DLITQLLRVL AIETKQEKAM AHADPTELAL SGLEAFSFDY IVKWPLSLII NRKALTRYQM LFRHMFYCKH VERQLCSVWI SNKTAKQHSL HSAQWFAGAF TLRQRMLNFV QNIQYYMMFE VMEPTWHILE KNLKSASNID DVLGHHTGFL DTCLKDCMLT NPELLKVFSK LMSVCVMFTN CMQKFTQSMK LDGELGGQTL EHSTVLGLPA GAEERARKEL ARKHLAEHAD TVQLVSGFEA TINKFDKNFS AHLLDLLARL SIYSTSDCEH GMASVISRLD FNGFYTERLE RLSAERSQKA TPQVPVLRGP PAPAPRVAVT AQGGGGGENL YFQGGGGGGS EELIKENMHM KLYMEGTVDN HHFKCTSEGE GKPYEGTQTM RIKVVEGGPL PFAFDILATS FLYGSKTFIN HTQGIPDFFK QSFPEGFTWE RVTTYEDGGV LTATQDTSLQ DGCLIYNVKI RGVNFTSNGP VMQKKTLGWE AFTETLYPAD GGLEGRNDMA LKLVGGSHLI ANIKTTYRSK KPAKNLKMPG VYYVDYRLER IKEANNETYV EQHEVAVARY CDLPSKLGHK LNGGGGGGLN DIFEAQKIEW HE

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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

BufferpH: 7.4
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 2400 / Average exposure time: 9.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. v.1.18)
Startup modelType of model: OTHER / Details: ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 522496

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