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- PDB-8ajb: Cryo-EM structure of crescentin filaments (stutter mutant, C2 sym... -

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Basic information

Entry
Database: PDB / ID: 8ajb
TitleCryo-EM structure of crescentin filaments (stutter mutant, C2 symmetry and large 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: 4.3 Å
AuthorsLiu, Y. / Lowe, J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust202754/Z/16/Z United Kingdom
Citation
Journal: 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.
#1: Journal: to be published
Title: Assembly and organization of the bacterial intermediate filament-like protein crescentin
Authors: Liu, Y. / Lowe, J.
History
DepositionJul 28, 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

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Assembly

Deposited unit
A: Crescentin
B: Crescentin
C: Crescentin
D: Crescentin
E: Crescentin-specific megabody MB13
F: Crescentin-specific megabody MB13
G: Crescentin
H: Crescentin
I: Crescentin
J: Crescentin
K: Crescentin-specific megabody MB13
L: Crescentin-specific megabody MB13
M: Crescentin
N: Crescentin
O: Crescentin
P: Crescentin
Q: Crescentin-specific megabody MB13
R: Crescentin-specific megabody MB13
S: Crescentin
T: Crescentin
U: Crescentin
V: Crescentin
W: Crescentin-specific megabody MB13
X: Crescentin-specific megabody MB13


Theoretical massNumber of molelcules
Total (without water)1,617,16624
Polymers1,617,16624
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area104940 Å2
ΔGint-667 kcal/mol
Surface area214330 Å2

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Components

#1: Protein
Crescentin /


Mass: 50177.816 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caulobacter vibrioides (bacteria) / Gene: creS, KZH45_19550 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A8F8EC09
#2: Antibody
Crescentin-specific megabody MB13


Mass: 101790.180 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelidae (mammal) / Production host: Escherichia coli BL21(DE3) (bacteria)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Crescentin filament in complex with megabody MB13COMPLEXall0RECOMBINANT
2CrescentinCOMPLEX#11RECOMBINANT
3Crescentin-specific megabody MB13COMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Caulobacter vibrioides (bacteria)155892
33Camelidae (mammal)9835
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 6.5 / Details: PIPES 25mM pH 6.5, 0.05% CHAPS
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: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 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: 2.4 sec. / Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
PDB_EXTRACT3.24data extraction
EM software
IDNameCategory
2EPUimage acquisition
4RELIONCTF correction
5cryoSPARCCTF correction
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13RELIONfinal Euler assignment
14cryoSPARCclassification
15cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 585252 / 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: 30 Å2 / Biso mean: 23.4573 Å2 / Biso min: 0.49 Å2

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