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- PDB-8fck: Structure of the vertebrate augmin complex -

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Basic information

Entry
Database: PDB / ID: 8fck
TitleStructure of the vertebrate augmin complex
Components
  • (HAUS augmin like complex subunit ...) x 4
  • (HAUS augmin-like complex subunit ...) x 4
KeywordsCELL CYCLE / microtubule / branching microtubule nucleation / spindle assembly
Function / homology
Function and homology information


HAUS complex / mitotic spindle microtubule / centrosome cycle / spindle assembly / bioluminescence / generation of precursor metabolites and energy / spindle / spindle pole / microtubule / cell division ...HAUS complex / mitotic spindle microtubule / centrosome cycle / spindle assembly / bioluminescence / generation of precursor metabolites and energy / spindle / spindle pole / microtubule / cell division / centrosome / cytoplasm
Similarity search - Function
HAUS augmin-like complex subunit 2, metazoa / HAUS augmin-like complex subunit 6 / HAUS augmin-like complex subunit 6, N-terminal / HAUS augmin-like complex subunit 2 / HAUS augmin-like complex subunit 7-like / HAUS augmin-like complex subunit 6 N-terminus / HAUS augmin-like complex subunit 2 / HAUS augmin-like complex subunit 4, metazoa / HAUS complex subunit 5, metazoa / HAUS augmin-like complex subunit 1 ...HAUS augmin-like complex subunit 2, metazoa / HAUS augmin-like complex subunit 6 / HAUS augmin-like complex subunit 6, N-terminal / HAUS augmin-like complex subunit 2 / HAUS augmin-like complex subunit 7-like / HAUS augmin-like complex subunit 6 N-terminus / HAUS augmin-like complex subunit 2 / HAUS augmin-like complex subunit 4, metazoa / HAUS complex subunit 5, metazoa / HAUS augmin-like complex subunit 1 / HAUS augmin-like complex subunit 5 / HAUS augmin-like complex subunit 4 / HAUS augmin-like complex subunit 4 / HAUS augmin-like complex subunit 5 / HAUS augmin-like complex subunit 3 / HAUS augmin-like complex subunit 3, N-terminal / HAUS augmin-like complex subunit 3 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
HAUS augmin-like complex subunit 5 / HAUS augmin-like complex subunit 1 / HAUS augmin like complex subunit 6 L homeolog / HAUS augmin like complex subunit 7 S homeolog / Green fluorescent protein / HAUS augmin-like complex subunit 8 / HAUS augmin like complex subunit 4 L homeolog / HAUS augmin-like complex subunit 3 / HAUS augmin like complex subunit 2 L homeolog
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.88 Å
AuthorsTravis, S.M. / Huang, W. / Zhang, R. / Petry, S.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM142149 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM138854 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM141100 United States
CitationJournal: Nat Commun / Year: 2023
Title: Integrated model of the vertebrate augmin complex.
Authors: Sophie M Travis / Brian P Mahon / Wei Huang / Meisheng Ma / Michael J Rale / Jodi Kraus / Derek J Taylor / Rui Zhang / Sabine Petry /
Abstract: Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high ...Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which rapidly amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but lack of structure information about augmin has hindered understanding how it promotes branching. In this work, we combine cryo-electron microscopy, protein structural prediction, and visualization of fused bulky tags via negative stain electron microscopy to identify the location and orientation of each subunit within the augmin structure. Evolutionary analysis shows that augmin's structure is highly conserved across eukaryotes, and that augmin contains a previously unidentified microtubule binding site. Thus, our findings provide insight into the mechanism of branching microtubule nucleation.
History
DepositionDec 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: HAUS augmin-like complex subunit 1
B: HAUS augmin-like complex subunit 3
C: HAUS augmin like complex subunit 4 L homeolog
D: HAUS augmin-like complex subunit 5
E: HAUS augmin like complex subunit 2 L homeolog, Green fluorescent protein chimera
F: HAUS augmin like complex subunit 6 L homeolog
G: HAUS augmin like complex subunit 7 S homeolog
H: HAUS augmin-like complex subunit 8


Theoretical massNumber of molelcules
Total (without water)404,3398
Polymers404,3398
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, All eight subunits coelute in a single fraction off gel filtration, as analyzed by SDS-PAGE
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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HAUS augmin-like complex subunit ... , 4 types, 4 molecules ABDH

#1: Protein HAUS augmin-like complex subunit 1


Mass: 32655.621 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: haus1.L / Plasmid: pFASTBAC / Cell (production host): Epithelial / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: A0A8J1L9M8
#2: Protein HAUS augmin-like complex subunit 3


Mass: 68112.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: haus3 / Plasmid: pFASTBAC / Cell (production host): Epithelial / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: Q6DCY9
#4: Protein HAUS augmin-like complex subunit 5


Mass: 77357.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: haus5.L / Plasmid: pFASTBAC / Cell (production host): Epithelial / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: A0A1L8FPI2
#8: Protein HAUS augmin-like complex subunit 8 / HEC1/NDC80-interacting centrosome-associated protein 1 / Sarcoma antigen NY-SAR-48 homolog


Mass: 41144.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: haus8, hice1 / Plasmid: pFASTBAC / Cell (production host): Epithelial / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: Q0IHJ3

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HAUS augmin like complex subunit ... , 4 types, 4 molecules CEFG

#3: Protein HAUS augmin like complex subunit 4 L homeolog / MGC115689 protein


Mass: 41256.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: haus4.L, haus4, MGC115689 / Plasmid: pFASTBAC / Cell (production host): Epithelial / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: Q4V7I1
#5: Protein HAUS augmin like complex subunit 2 L homeolog, Green fluorescent protein chimera / MGC82377 protein


Mass: 53505.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: haus2.L, cep27, haus2, MGC82377, GFP / Plasmid: pFASTBAC / Cell (production host): Epithelial / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: Q6INL9, UniProt: P42212
#6: Protein HAUS augmin like complex subunit 6 L homeolog


Mass: 50927.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: haus6.L / Plasmid: pFASTBAC / Cell (production host): Epithelial / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: A0JPI0
#7: Protein HAUS augmin like complex subunit 7 S homeolog / LOC100158301 protein


Mass: 39379.605 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: haus7.S, haus7, LOC100158301, uchl5ip, uip1 / Plasmid: pFASTBAC / Cell (production host): Epithelial / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: B1H1T5

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Augmin / Type: COMPLEX / Details: Peak octameric fraction following gel filtration / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.403 MDa / Experimental value: NO
Source (natural)Organism: Xenopus laevis (African clawed frog) / Cellular location: cytoplasm / Organelle: mitotic spindle
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9 / Plasmid: pFASTBAC
Buffer solutionpH: 7.7
Buffer component
IDConc.NameBuffer-ID
110 mMHEPES1
2100 mMKCl1
35 mMEGTA1
42 mMMgCl21
51 mMDTT1
60.05 % (v/v)NP-401
SpecimenConc.: 0.06 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Nominal defocus max: 39903 nm / Nominal defocus min: 10105 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2350

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.3.2+220824particle selectioncryoSPARC template picker was used to select particle images based on negative stain classes
2cryoSPARCimage acquisition
4cryoSPARCv3.3.2+220824CTF correctionCryoSparc PatchCTF was used for image correction
7UCSF ChimeraX1.4model fittingfitmap was used to rigid-body fit model into map
10cryoSPARCv3.3.2+220824initial Euler assignment
11cryoSPARCv3.3.2+220824final Euler assignment
12cryoSPARCv3.3.2+220824classification
13cryoSPARCv3.3.2+2208243D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2046060
Details: Template picks, trained on evenly-spaced templates from ab initio model
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00125535
ELECTRON MICROSCOPYf_angle_d0.35334381
ELECTRON MICROSCOPYf_dihedral_angle_d8.6179956
ELECTRON MICROSCOPYf_chiral_restr0.0293872
ELECTRON MICROSCOPYf_plane_restr0.0024458

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