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- EMDB-22874: Cryo-electron tomography and subtomographic averaging reveals cil... -

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Basic information

Entry
Database: EMDB / ID: EMD-22874
TitleCryo-electron tomography and subtomographic averaging reveals ciliary defects underlying human RSPH1 primary ciliary dyskinesia
Map data
Sample
  • Organelle or cellular component: respiratory ciliary axonemes of human with RSPH1 primary ciliary dyskinesia disease
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 33.6 Å
AuthorsZhao Y / Pinskey J / Lin J / Yin W / Leigh MW / Zariwala MA / Knowles MR / Ostrowski LE / Nicastro D
Funding support United States, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM083122 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM137470 United States
Cystic Fibrosis FoundationR026-CR11 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DK065988 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)HL117836 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)HL096458 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)HL071798 United States
CitationJournal: Mol Biol Cell / Year: 2021
Title: Structural insights into the cause of human primary ciliary dyskinesia.
Authors: Yanhe Zhao / Justine Pinskey / Jianfeng Lin / Weining Yin / Patrick R Sears / Leigh A Daniels / Maimoona A Zariwala / Michael R Knowles / Lawrence E Ostrowski / Daniela Nicastro /
Abstract: Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A () can lead to primary ciliary ...Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A () can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of PCD remains elusive. Here, we present the native three-dimensional structure of human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in cilia. However, cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination.
History
DepositionOct 19, 2020-
Header (metadata) releaseMay 5, 2021-
Map releaseMay 5, 2021-
UpdateJun 9, 2021-
Current statusJun 9, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 131
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 131
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22874.png

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Map

FileDownload / File: emd_22874.map.gz / Format: CCP4 / Size: 4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 10 Å
Density
Contour LevelBy AUTHOR: 131.0 / Movie #1: 131
Minimum - Maximum90.804886 - 168.5636
Average (Standard dev.)126.77934 (±6.4328465)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-250-34
Dimensions8614086
Spacing1408686
CellA: 1400.0 Å / B: 860.0 Å / C: 860.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z101010
M x/y/z1408686
origin x/y/z0.0000.0000.000
length x/y/z1400.000860.000860.000
α/β/γ90.00090.00090.000
start NX/NY/NZ727265
NX/NY/NZ157157169
MAP C/R/S123
start NC/NR/NS0-25-34
NC/NR/NS1408686
D min/max/mean90.805168.564126.779

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Supplemental data

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Sample components

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Entire : respiratory ciliary axonemes of human with RSPH1 primary ciliary ...

EntireName: respiratory ciliary axonemes of human with RSPH1 primary ciliary dyskinesia disease
Components
  • Organelle or cellular component: respiratory ciliary axonemes of human with RSPH1 primary ciliary dyskinesia disease

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Supramolecule #1: respiratory ciliary axonemes of human with RSPH1 primary ciliary ...

SupramoleculeName: respiratory ciliary axonemes of human with RSPH1 primary ciliary dyskinesia disease
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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: 7.3
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average electron dose: 1.8 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 20 / Number images used: 3044
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 33.6 Å / Resolution method: FSC 0.5 CUT-OFF / Number subtomograms used: 3044

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