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- PDB-9dsm: Cryo-EM structure of SSNA-1(R18E/R20E/Q98E) filaments -

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Basic information

Entry
Database: PDB / ID: 9dsm
TitleCryo-EM structure of SSNA-1(R18E/R20E/Q98E) filaments
ComponentsSjogren's Syndrome Nuclear Autoantigen
KeywordsCELL CYCLE / SSNA1 / DIP13 / NA14 / microtubules / centriole / centrosome / mitotic spindle / cytoskeleton / coiled-coil
Function / homologySjogren's Syndrome Nuclear Autoantigen
Function and homology information
Biological speciesCaenorhabditis elegans (invertebrata)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.55 Å
AuthorsAgostini, L. / Biertumpfel, C. / Mizuno, N.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural insights into SSNA1 self-assembly and its microtubule binding for centriole maintenance.
Authors: Lorenzo Agostini / Jason A Pfister / Nirakar Basnet / Jienyu Ding / Rui Zhang / Christian Biertümpfel / Kevin F O'Connell / Naoko Mizuno /
Abstract: SSNA1 is a fibrillar protein involved in dynamic microtubule remodeling, including nucleation, co-polymerization, and microtubule branching. The underlying molecular mechanism has remained unclear ...SSNA1 is a fibrillar protein involved in dynamic microtubule remodeling, including nucleation, co-polymerization, and microtubule branching. The underlying molecular mechanism has remained unclear due to a lack of structural information. Here, we determine the cryo-EM structure of C.elegans SSNA-1 at 4.55-Å resolution and evaluate its role in embryonic development. We find that SSNA-1 forms an anti-parallel coiled-coil, with self-assembly facilitated by an overhang of 16 C-terminal residues that form a triple-stranded helical junction. The microtubule-binding region is within the triple-stranded junction, suggesting that self-assembly of SSNA-1 creates hubs for effective microtubule interaction. Genetical analysis elucidates that SSNA-1 deletion significantly reduces embryonic viability, and causes multipolar spindles during cell division. Interestingly, impairing SSNA-1 self-assembly has a comparable effect on embryonic viability as the knockout strain. Our study provides molecular insights into SSNA-1's self-assembly and its role in microtubule binding and cell division regulation through centriole stability.
History
DepositionSep 27, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 27, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sjogren's Syndrome Nuclear Autoantigen
B: Sjogren's Syndrome Nuclear Autoantigen
C: Sjogren's Syndrome Nuclear Autoantigen
D: Sjogren's Syndrome Nuclear Autoantigen
E: Sjogren's Syndrome Nuclear Autoantigen
F: Sjogren's Syndrome Nuclear Autoantigen
G: Sjogren's Syndrome Nuclear Autoantigen
H: Sjogren's Syndrome Nuclear Autoantigen
I: Sjogren's Syndrome Nuclear Autoantigen
J: Sjogren's Syndrome Nuclear Autoantigen
K: Sjogren's Syndrome Nuclear Autoantigen
L: Sjogren's Syndrome Nuclear Autoantigen
M: Sjogren's Syndrome Nuclear Autoantigen
N: Sjogren's Syndrome Nuclear Autoantigen
O: Sjogren's Syndrome Nuclear Autoantigen
P: Sjogren's Syndrome Nuclear Autoantigen
Q: Sjogren's Syndrome Nuclear Autoantigen
R: Sjogren's Syndrome Nuclear Autoantigen
S: Sjogren's Syndrome Nuclear Autoantigen
T: Sjogren's Syndrome Nuclear Autoantigen
U: Sjogren's Syndrome Nuclear Autoantigen
V: Sjogren's Syndrome Nuclear Autoantigen
W: Sjogren's Syndrome Nuclear Autoantigen
X: Sjogren's Syndrome Nuclear Autoantigen
Y: Sjogren's Syndrome Nuclear Autoantigen
Z: Sjogren's Syndrome Nuclear Autoantigen
a: Sjogren's Syndrome Nuclear Autoantigen
b: Sjogren's Syndrome Nuclear Autoantigen
c: Sjogren's Syndrome Nuclear Autoantigen
d: Sjogren's Syndrome Nuclear Autoantigen
e: Sjogren's Syndrome Nuclear Autoantigen
f: Sjogren's Syndrome Nuclear Autoantigen


Theoretical massNumber of molelcules
Total (without water)469,32132
Polymers469,32132
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, negative stain filaments, light scattering, dynamic light-scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ...
Sjogren's Syndrome Nuclear Autoantigen


Mass: 14666.279 Da / Num. of mol.: 32 / Mutation: R18E,R20E,Q98E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: ssna-1, tag-261, CELE_T07A9.13, T07A9.13 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): gold / References: UniProt: Q5F4U5
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: self-assembled SSNA-1 filaments / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 20 kDa/nm / Experimental value: YES
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)gold
Buffer solutionpH: 7.5
Details: 50 mM sodium phosphate buffer-NaOH pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 1 mM DTT, 0.1% (v/v) n-octyl-beta-D-glucopyranoside
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium phosphateNaH2PO41
210 mMsodium hydroxideNaOH1
3150 mMsodium chlorideNaCl1
410 %glycerolHOCH2CH(OH)CH2OH1
51 mMDTTHSCH2CH(OH)CH(OH)CH2SH1
60.1 %n-octyl-beta-D-glucopyranosideC14H28O61
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: SSNA-1(R18E/R20E/Q98E) was dialyzed overnight at 4degC against buffer to grow filaments. 5 ul sample at a final concentration of 0.1 mg/mL was applied to glow discharged Quantifoil grids R2/1.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 288 K
Details: After 1 min incubation at room temperature, the grid was manually blotted on the side of the grid with filter paper (Whatman Grade 1, Cytiva #1001-055) and blotted again with 4 uL of a ...Details: After 1 min incubation at room temperature, the grid was manually blotted on the side of the grid with filter paper (Whatman Grade 1, Cytiva #1001-055) and blotted again with 4 uL of a solution containing lysis buffer supplemented with 0.1 %(v/v) n-octyl-beta-D-glucopyranoside (beta-OG, Anatrace #O311). The grid was then transferred to an EM GP2 plunger (Leica) with a chamber temperature of 15degC, maximal humidity, incubated for 1 min, blotted with Grade 595 filter paper (Ted Pella #47000-200) for 6 s and vitrified in liquid 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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 40.84 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8390 / Details: pixel size 0.412
EM imaging opticsDetails: pixel size 0.412

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.3particle selection
2EPUimage acquisition
4cryoSPARC4.5.3CTF correction
7Coot0.9.6model fitting
9PHENIX1.21_5207+SVNmodel refinement
10cryoSPARC4.5.3initial Euler assignment
11cryoSPARC4.5.3final Euler assignment
12cryoSPARC4.5.3classification
13cryoSPARC4.5.33D reconstruction
CTF correctionType: NONE
Helical symmertyAngular rotation/subunit: 45 ° / Axial rise/subunit: 112 Å / Axial symmetry: C8
Particle selectionNum. of particles selected: 888099
3D reconstructionResolution: 4.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80664 / Algorithm: BACK PROJECTION / Details: no helical parameters used / Symmetry type: HELICAL
Atomic model buildingB value: 265.8 / Protocol: OTHER / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00226720
ELECTRON MICROSCOPYf_angle_d0.42835616
ELECTRON MICROSCOPYf_dihedral_angle_d5.5773584
ELECTRON MICROSCOPYf_chiral_restr0.0283840
ELECTRON MICROSCOPYf_plane_restr0.0024736

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