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- EMDB-41317: CryoET reconstruction of 48-nm repeat doublet microtubule from hu... -

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Entry
Database: EMDB / ID: EMD-41317
TitleCryoET reconstruction of 48-nm repeat doublet microtubule from human sperm
Map dataComposite map for the human dmt48nm combining register 1 and 2. (correct handiness)
Sample
  • Complex: Microtubule doublets from mouse sperm flagella treated by EHNA
KeywordsMammalian sperm / axoneme / microtubule-based structure / microtubule inner protein / non-motor proteins / cellular motility / fertility / structural protein
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 10.4 Å
AuthorsChen Z / Shiozak M / Hass KM / Skinner W / Zhao S / Guo C / Polacco BJ / Yu Z / Krogan NJ / Kaake RM ...Chen Z / Shiozak M / Hass KM / Skinner W / Zhao S / Guo C / Polacco BJ / Yu Z / Krogan NJ / Kaake RM / Vale RD / Agard DA
Funding support United States, 3 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118106 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118099 United States
CitationJournal: Cell / Year: 2023
Title: De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.
Authors: Zhen Chen / Momoko Shiozaki / Kelsey M Haas / Will M Skinner / Shumei Zhao / Caiying Guo / Benjamin J Polacco / Zhiheng Yu / Nevan J Krogan / Polina V Lishko / Robyn M Kaake / Ronald D Vale / David A Agard /
Abstract: To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their ...To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.
History
DepositionJul 24, 2023-
Header (metadata) releaseNov 8, 2023-
Map releaseNov 8, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41317.png

Downloads & links

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Map

FileDownload / File: emd_41317.map.gz / Format: CCP4 / Size: 80.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map for the human dmt48nm combining register 1 and 2. (correct handiness)
Voxel sizeX=Y=Z: 2.65 Å
Density
Contour LevelBy AUTHOR: 0.0045
Minimum - Maximum-0.006505797 - 0.012322627
Average (Standard dev.)0.00040487363 (±0.0014558171)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions276276276
Spacing276276276
CellA=B=C: 731.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41317_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Mask for the register 2 of dmt48 (correct handiness)

Fileemd_41317_additional_1.map
AnnotationMask for the register 2 of dmt48 (correct handiness)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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Additional map: Half map1 for the register 2 of dmt48 (correct handiness)

Fileemd_41317_additional_2.map
AnnotationHalf map1 for the register 2 of dmt48 (correct handiness)
Projections & Slices
AxesZYX

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Histogram

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Additional map: Half map2 for the register 2 of dmt48 (correct handiness)

Fileemd_41317_additional_3.map
AnnotationHalf map2 for the register 2 of dmt48 (correct handiness)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map1 for the register 1 of dmt48 (correct handiness)

Fileemd_41317_half_map_1.map
AnnotationHalf map1 for the register 1 of dmt48 (correct handiness)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map2 for the register 1 of dmt48 (correct handiness)

Fileemd_41317_half_map_2.map
AnnotationHalf map2 for the register 1 of dmt48 (correct handiness)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Microtubule doublets from mouse sperm flagella treated by EHNA

EntireName: Microtubule doublets from mouse sperm flagella treated by EHNA
Components
  • Complex: Microtubule doublets from mouse sperm flagella treated by EHNA

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Supramolecule #1: Microtubule doublets from mouse sperm flagella treated by EHNA

SupramoleculeName: Microtubule doublets from mouse sperm flagella treated by EHNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#35
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57Bl/6J
Molecular weightTheoretical: 30 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 4.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 2.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0-beta2) / Number subtomograms used: 11358
ExtractionNumber tomograms: 50 / Number images used: 11358
Final angle assignmentType: MAXIMUM LIKELIHOOD

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