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- PDB-8afl: Cryo-EM structure of crescentin filaments (wildtype, C1 symmetry ... -

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Basic information

Entry
Database: PDB / ID: 8afl
TitleCryo-EM structure of crescentin filaments (wildtype, C1 symmetry and small box)
Components
  • Crescentin-specific megabody MB13
  • Crescentin
KeywordsSTRUCTURAL PROTEIN / cytoskeleton / cell shape / intermediate filaments / coiled coil / assembly
Function / homology:
Function and homology information
Biological speciesCaulobacter vibrioides (bacteria)
Camelidae (mammal)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsLiu, Y. / Lowe, J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust202754/Z/16/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Filament structure and subcellular organization of the bacterial intermediate filament-like protein crescentin.
Authors: Yue Liu / Fusinita van den Ent / Jan Löwe /
Abstract: The protein crescentin is required for the crescent shape of the freshwater bacterium (). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features ...The protein crescentin is required for the crescent shape of the freshwater bacterium (). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, including the formation of static filaments based on long and parallel coiled coils, the protein's length, structural roles in cell and organelle shape determination and the presence of a coiled coil discontinuity called the "stutter." Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by twofold symmetry, that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine cross-linking, we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. When comparing with current models of IF proteins and their filaments, which are also built from parallel coiled coil dimers and lack overall polarity, it emerges that IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures.
History
DepositionJul 18, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Crescentin
B: Crescentin
D: Crescentin-specific megabody MB13
C: Crescentin-specific megabody MB13
E: Crescentin
F: Crescentin


Theoretical massNumber of molelcules
Total (without water)403,2556
Polymers403,2556
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Crescentin /


Mass: 49918.559 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caulobacter vibrioides (bacteria) / Gene: creS, KZH45_19550 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8F8EC09
#2: Antibody Crescentin-specific megabody MB13


Mass: 101790.180 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelidae (mammal) / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Crescentin filaments in complex with megabodies / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Caulobacter vibrioides (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.5 / Details: PIPES 25mM, 0.05% CHAPS, pH 6.5
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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 FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 34 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2

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Processing

Software
NameVersionClassificationNB
PHENIX(1.18.2_3874:phenix.real_space_refine)refinement
PDB_EXTRACT3.24data extraction
EM software
IDNameCategory
1Topazparticle selection
2EPUimage acquisition
4RELIONCTF correction
5cryoSPARCCTF correction
8UCSF Chimeramodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13RELIONclassification
14cryoSPARC3D reconstruction
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1101149 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 115.76 Å2 / Biso mean: 52.8292 Å2 / Biso min: 21.56 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044106
ELECTRON MICROSCOPYf_angle_d0.6555520
ELECTRON MICROSCOPYf_chiral_restr0.041624
ELECTRON MICROSCOPYf_plane_restr0.003738
ELECTRON MICROSCOPYf_dihedral_angle_d19.24588

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