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- PDB-5flz: Cryo-EM structure of gamma-TuSC oligomers in a closed conformation -

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Entry
Database: PDB / ID: 5flz
TitleCryo-EM structure of gamma-TuSC oligomers in a closed conformation
Components
  • SPINDLE POLE BODY COMPONENT 110
  • SPINDLE POLE BODY COMPONENT SPC97
  • SPINDLE POLE BODY COMPONENT SPC98
  • TUBULIN GAMMA CHAIN
KeywordsCELL CYCLE / MICROTUBULE NUCLEATION
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 / meiotic spindle organization / gamma-tubulin complex / 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 / meiotic spindle organization / gamma-tubulin complex / microtubule nucleation / positive regulation of cytoplasmic translation / gamma-tubulin binding / spindle pole body / mitotic sister chromatid segregation / spindle assembly / cytoplasmic microtubule organization / mitotic spindle organization / meiotic cell cycle / structural constituent of cytoskeleton / spindle pole / spindle / 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: 6.9 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: Nat Struct Mol Biol / Year: 2015
Title: Ring closure activates yeast γTuRC for species-specific microtubule nucleation.
Authors: Justin M Kollman / Charles H Greenberg / Sam Li / Michelle Moritz / Alex Zelter / Kimberly K Fong / Jose-Jesus Fernandez / Andrej Sali / John Kilmartin / Trisha N Davis / David A Agard /
Abstract: The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-tubulin small complex (γTuSC) subunits and is thought to function as a ...The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm the functional importance of the closed γTuSC ring, we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, thus suggesting that this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a strong preference for tubulin from the same species.
History
DepositionOct 29, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 13, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 23, 2016Group: Database references / Other
Revision 1.2May 11, 2016Group: Database references / Other
Revision 1.3Aug 2, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 1.4May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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)308,2696
Polymers308,2696
Non-polymers00
Water00
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)2,157,88142
Polymers2,157,88142
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)
Production host: UNIDENTIFIED BACULOVIRUS / 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)
Production host: UNIDENTIFIED BACULOVIRUS / References: UniProt: P53540
#3: Protein TUBULIN GAMMA CHAIN / GAMMA-TUBULIN


Mass: 52733.340 Da / Num. of mol.: 2 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: CYSTEINE RESIDUES WERE INTRODUCED AT POSITIONS 58 AND 288 TO PROMOTE CROSSLINKING OF THE HELICAL COMPLEX
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Production host: UNIDENTIFIED BACULOVIRUS / 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: RESIDUES 1-220 OF SPC110 WERE EXPRESSED WITH AN N-TERMINAL GST TAG, WHICH WAS CLEAVED OFF DURING PURIFICATION. IT CANNOT BE UNIQUELY IDENTIFIED WHICH FRAGMENT OF SPC110 IS PRESENT IN THE ...Details: RESIDUES 1-220 OF SPC110 WERE EXPRESSED WITH AN N-TERMINAL GST TAG, WHICH WAS CLEAVED OFF DURING PURIFICATION. IT CANNOT BE UNIQUELY IDENTIFIED WHICH FRAGMENT OF SPC110 IS PRESENT IN THE MAP, THUS A POLY-ALANINE HELIX WAS FITTED INTO THE MAP.
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Production host: UNIDENTIFIED BACULOVIRUS
Sequence detailsCONTAINS TWO MUTATIONS TO FORCE CLOSURE, S58C AND G288C 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: RECOMBINANT YEAST GAMMA- TUSC MUTANT S58C G288C / Type: COMPLEX
Buffer solutionName: 40 MM HEPES PH 7.6, 100 MM KCL, 1 MM EGTA, 1MM MGCL2, 1 MM OXIDIZED GLUTATHIONE
pH: 7.6
Details: 40 MM HEPES PH 7.6, 100 MM KCL, 1 MM EGTA, 1MM MGCL2, 1 MM OXIDIZED GLUTATHIONE
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: CRYOGEN- ETHANE, HUMIDITY- 90, INSTRUMENT- FEI VITROBOT MARK I, METHOD- BLOT FOR 2-5 SECONDS BEFORE PLUNGING,

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: May 25, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 94000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.12 mm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k)
Image scansNum. digital images: 364

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Processing

EM software
IDNameVersionCategoryDetails
1CTFFINDCTF correction
2MDFFmodel fitting
3MODELLERmodel fitting
4EMAN13D reconstruction
5SPIDER3D reconstruction
6hsearch_lorentzsymmetry determinationhelical symmetry parameter refinement
CTF correctionDetails: EACH MICROGRAPH
3D reconstructionMethod: REFERENCE MATCHING / Resolution: 6.9 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.COM/INTEGRATIVEMODELING/GAMMA-TUSC SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2799. (DEPOSITION ID: 12869).
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: METHOD--FLEXIBLE FITTING
RefinementHighest resolution: 6.9 Å
Refinement stepCycle: LAST / Highest resolution: 6.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16942 0 0 0 16942

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