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- PDB-5fm1: Structure of gamma-tubulin small complex based on a cryo-EM map, ... -

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Basic information

Entry
Database: PDB / ID: 5fm1
TitleStructure of gamma-tubulin small complex based on a cryo-EM map, chemical cross-links, and a remotely related structure
Components
  • SPINDLE POLE BODY COMPONENT 110
  • SPINDLE POLE BODY COMPONENT SPC97
  • SPINDLE POLE BODY COMPONENT SPC98
  • TUBULIN GAMMA CHAIN
KeywordsCELL CYCLE / MICROTUBULE / NUCLEATION / TUBULIN / FILAMENT
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 / cytoplasmic microtubule organization / spindle assembly / 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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 8 Å
AuthorsGreenberg, C.H. / Kollman, J. / Zelter, A. / Johnson, R. / MacCoss, M.J. / Davis, T.N. / Agard, D.A. / Sali, A.
Citation
Journal: J Struct Biol / Year: 2016
Title: Structure of γ-tubulin small complex based on a cryo-EM map, chemical cross-links, and a remotely related structure.
Authors: Charles H Greenberg / Justin Kollman / Alex Zelter / Richard Johnson / Michael J MacCoss / Trisha N Davis / David A Agard / Andrej Sali /
Abstract: Modeling protein complex structures based on distantly related homologues can be challenging due to poor sequence and structure conservation. Therefore, utilizing even low-resolution experimental ...Modeling protein complex structures based on distantly related homologues can be challenging due to poor sequence and structure conservation. Therefore, utilizing even low-resolution experimental data can significantly increase model precision and accuracy. Here, we present models of the two key functional states of the yeast γ-tubulin small complex (γTuSC): one for the low-activity "open" state and another for the higher-activity "closed" state. Both models were computed based on remotely related template structures and cryo-EM density maps at 6.9Å and 8.0Å resolution, respectively. For each state, extensive sampling of alignments and conformations was guided by the fit to the corresponding cryo-EM density map. The resulting good-scoring models formed a tightly clustered ensemble of conformations in most regions. We found significant structural differences between the two states, primarily in the γ-tubulin subunit regions where the microtubule binds. We also report a set of chemical cross-links that were found to be consistent with equilibrium between the open and closed states. The protocols developed here have been incorporated into our open-source Integrative Modeling Platform (IMP) software package (http://integrativemodeling.org), and can therefore be applied to many other systems.
#1: Journal: Nature / Year: 2010
Title: Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.
Authors: Justin M Kollman / Jessica K Polka / Alex Zelter / Trisha N Davis / David A Agard /
Abstract: Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory ...Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.
History
DepositionOct 30, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 3, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 23, 2016Group: Database references
Revision 1.2May 11, 2016Group: Database references / Other
Revision 1.3Aug 23, 2017Group: Data collection / Category: em_image_scans / em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Assembly

Deposited unit
A: SPINDLE POLE BODY COMPONENT SPC97
B: SPINDLE POLE BODY COMPONENT SPC98
C: TUBULIN GAMMA CHAIN
D: TUBULIN GAMMA CHAIN
E: SPINDLE POLE BODY COMPONENT 110
F: SPINDLE POLE BODY COMPONENT 110


Theoretical massNumber of molelcules
Total (without water)308,1446
Polymers308,1446
Non-polymers00
Water0
1
A: SPINDLE POLE BODY COMPONENT SPC97
B: SPINDLE POLE BODY COMPONENT SPC98
C: TUBULIN GAMMA CHAIN
D: TUBULIN GAMMA CHAIN
E: SPINDLE POLE BODY COMPONENT 110
F: SPINDLE POLE BODY COMPONENT 110
x 7


Theoretical massNumber of molelcules
Total (without water)2,157,01142
Polymers2,157,01142
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
point symmetry operation6
Number of models10

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Components

#1: Protein SPINDLE POLE BODY COMPONENT SPC97 / / GCP2


Mass: 96940.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PDV45 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: P38863
#2: Protein SPINDLE POLE BODY COMPONENT SPC98 / / GCP3


Mass: 98336.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PDV45 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: P53540
#3: Protein TUBULIN GAMMA CHAIN / GAMMA-TUBULIN


Mass: 52671.188 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PDV45 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: P53378
#4: Protein/peptide SPINDLE POLE BODY COMPONENT 110 / / EXTRAGENIC SUPPRESSOR OF CMD1-1 MUTANT PROTEIN 1 / NUCLEAR FILAMENT-RELATED PROTEIN 1 / SPINDLE ...EXTRAGENIC SUPPRESSOR OF CMD1-1 MUTANT PROTEIN 1 / NUCLEAR FILAMENT-RELATED PROTEIN 1 / SPINDLE POLE BODY SPACER PROTEIN SPC110 / SPC110


Mass: 3762.629 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: CONSTRUCT INCLUDES THE FIRST 220 RESIDUES OF SPC110P IT CANNOT BE UNIQUELY IDENTIFIED WHICH FRAGMENT OF SPC110 IS PRESENT IN THE MAP, THUS A POLY-ALANINE HELIX WAS FITTED.
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PDV45 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm)
Sequence detailsONLY RESIDUES 1-220 INCLUDED IN STRUCTURE. CHAINS E AND F CORRESPOND TO UNIPROT ENTRY P32380

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: GAMMA TUBULIN SMALL COMPLEX / Type: COMPLEX
Buffer solutionName: 100 MM KCL, 1MM GTP, 1MM MGCL2, 2MM EGTA, 40 MM HEPES / pH: 7.6
Details: 100 MM KCL, 1MM GTP, 1MM MGCL2, 2MM EGTA, 40 MM HEPES
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: OTHER
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Sep 23, 2009
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 60000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm / Cs: 2.2 mm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k)

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Processing

EM software
IDNameCategory
1IMPmodel fitting
2MDFFmodel fitting
3MODELLERmodel fitting
4SPIDER3D reconstruction
CTF correctionDetails: WHOLE MICROGRAPH
3D reconstructionMethod: ITERATIVE HELICAL REAL SPACE RECONSTRUCTION / Resolution: 8 Å
Details: INITIAL MODELS WERE GENERATED WITH MODELER 9.15 BASED ON PDB ENTRIES 3RIP (FOR GCP2 AND GCP3) AND 3CB2 (FOR GAMMA TUBULIN). TWO COMPLETE GAMMA-TUSC STRUCTURES WERE MODELED SIMULTANEOUSLY, ...Details: INITIAL MODELS WERE GENERATED WITH MODELER 9.15 BASED ON PDB ENTRIES 3RIP (FOR GCP2 AND GCP3) AND 3CB2 (FOR GAMMA TUBULIN). TWO COMPLETE GAMMA-TUSC STRUCTURES WERE MODELED SIMULTANEOUSLY, RIGIDLY FITTED INTO THE CRYO-EM MAP WITH UCSF CHIMERA, THEN FLEXIBLY FITTED WITH MDFF. ADDITIONAL SECONDARY STRUCTURE AND SYMMETRY RESTRAINTS WERE ADDED. FINAL VALIDATION PERFORMED WITH THE INTEGRATIVE MODELING PLATFORM. FOR DETAILS, ALL CODE IS DEPOSITED AT GITHUB. COMSLASHINTEGRATIVEMODELINGSLASHGAMMA-TUSC SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1731. (DEPOSITION ID: 7273).
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: METHOD--FLEXIBLE FITTING REFINEMENT PROTOCOL--X-RAY
RefinementHighest resolution: 8 Å
Refinement stepCycle: LAST / Highest resolution: 8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16938 0 0 0 16938

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