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- EMDB-20637: Composite cryo-EM density map of the 48-nm repeat of the doublet ... -

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Basic information

Entry
Database: EMDB / ID: EMD-20637
TitleComposite cryo-EM density map of the 48-nm repeat of the doublet microtubule from Chlamydomonas reinhardtii fap166 mutant
Map dataComposite cryo-EM density map of the 48-nm repeat of the doublet microtubule from Chlamydomonas reinhardtii fap166 mutant
Sample
  • Complex: doublet microtubule from Chlamydomonas reinhardtii fap166 mutant
Biological speciesChlamydomonas reinhardtii (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsMa M / Stoyanova M / Rademacher G / Dutcher SK / Brown A / Zhang R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01GM032843 United States
CitationJournal: Cell / Year: 2019
Title: Structure of the Decorated Ciliary Doublet Microtubule.
Authors: Meisheng Ma / Mihaela Stoyanova / Griffin Rademacher / Susan K Dutcher / Alan Brown / Rui Zhang /
Abstract: The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility ...The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.
History
DepositionAug 22, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseNov 13, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20637.png

Downloads & links

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Map

FileDownload / File: emd_20637.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite cryo-EM density map of the 48-nm repeat of the doublet microtubule from Chlamydomonas reinhardtii fap166 mutant
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 512 pix.
= 718.336 Å		1.4 Å/pix.
x 512 pix.
= 718.336 Å		1.4 Å/pix.
x 512 pix.
= 718.336 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.403 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.027772339 - 0.09814696
Average (Standard dev.)0.0027186193 (±0.006375605)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 718.336 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.4031.4031.403
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z718.336718.336718.336
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0280.0980.003

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Supplemental data

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Mask #1

Fileemd_20637_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_20637_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_20637_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : doublet microtubule from Chlamydomonas reinhardtii fap166 mutant

EntireName: doublet microtubule from Chlamydomonas reinhardtii fap166 mutant
Components
  • Complex: doublet microtubule from Chlamydomonas reinhardtii fap166 mutant

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Supramolecule #1: doublet microtubule from Chlamydomonas reinhardtii fap166 mutant

SupramoleculeName: doublet microtubule from Chlamydomonas reinhardtii fap166 mutant
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: fap166

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4 / Component - Name: HMDEKP
Details: 30 mM HEPES, 5 mM MgSO4, 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, PH 7.4
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Microscope is equipped with a Cs corrector
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 6 / Number real images: 8314 / Average exposure time: 9.0 sec. / Average electron dose: 38.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.1 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe movies were drift-corrected using UCSF MotionCorr2
Particle selectionDetails: doublet microtubules were manually selected from the raw micrographs
CTF correctionSoftware: (Name: Gctf, RELION)
Details: CTF parameters were estimated using Gctf and corrected during 3D reconstruction within RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 19800
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 300
Projection matching processing - Angular sampling: 4.0 degrees
Software - Name: EMAN
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final 3D classificationNumber classes: 6 / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 20

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