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- EMDB-41451: Focused reconstruction of doublets from mouse sperm (register #2) -

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Basic information

Entry
Database: EMDB / ID: EMD-41451
TitleFocused reconstruction of doublets from mouse sperm (register #2)
Map data
Sample
  • Cell: Mouse sperm
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: 7.7 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
DepositionAug 3, 2023-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41451.png

Downloads & links

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Map

FileDownload / File: emd_41451.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.65 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.14950773 - 0.25261417
Average (Standard dev.)0.0020904525 (±0.025631687)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 583.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41451_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41451_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41451_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mouse sperm

EntireName: Mouse sperm
Components
  • Cell: Mouse sperm

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Supramolecule #1: Mouse sperm

SupramoleculeName: Mouse sperm / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)

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

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Image processing

ExtractionNumber tomograms: 76 / Number images used: 32288
Details: 32288 particles were initially picked every 24 nm along the microtubules.
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0-beta2) / Number subtomograms used: 12848

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