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- PDB-5z1l: Cryo-EM structure of Methanoccus maripaludis archaellum -

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Basic information

Entry
Database: PDB / ID: 5z1l
TitleCryo-EM structure of Methanoccus maripaludis archaellum
ComponentsFlagellin
KeywordsPROTEIN FIBRIL / Archaellum / archea / flagellum / metal binding / motility / helical
Function / homologyarchaeal-type flagellum / Flagellin, archaea / Archaebacterial flagellin / Flagellin/pilin, N-terminal / archaeal or bacterial-type flagellum-dependent cell motility / structural molecule activity / membrane / Flagellin
Function and homology information
Biological speciesMethanococcus maripaludis (archaea)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4 Å
AuthorsMeshcheryakov, V.A. / Shibata, S. / Schreiber, M.T. / Villar-Briones, A. / Jarrell, K.F. / Aizawa, S. / Wolf, M.
Funding support Japan, 2items
OrganizationGrant numberCountry
JSPS Kakenhi17K17085 Japan
SPRING8Proposal No. 2014B1341 Japan
CitationJournal: EMBO Rep / Year: 2019
Title: High-resolution archaellum structure reveals a conserved metal-binding site.
Authors: Vladimir A Meshcheryakov / Satoshi Shibata / Makoto Tokoro Schreiber / Alejandro Villar-Briones / Kenneth F Jarrell / Shin-Ichi Aizawa / Matthias Wolf /
Abstract: Many archaea swim by means of archaella. While the archaellum is similar in function to its bacterial counterpart, its structure, composition, and evolution are fundamentally different. Archaella are ...Many archaea swim by means of archaella. While the archaellum is similar in function to its bacterial counterpart, its structure, composition, and evolution are fundamentally different. Archaella are related to archaeal and bacterial type IV pili. Despite recent advances, our understanding of molecular processes governing archaellum assembly and stability is still incomplete. Here, we determine the structures of archaella by X-ray crystallography and cryo-EM The crystal structure of FlaB1 is the first and only crystal structure of any archaellin to date at a resolution of 1.5 Å, which is put into biological context by a cryo-EM reconstruction from archaella at 4 Å resolution created with helical single-particle analysis. Our results indicate that the archaellum is predominantly composed of FlaB1. We identify N-linked glycosylation by cryo-EM and mass spectrometry. The crystal structure reveals a highly conserved metal-binding site, which is validated by mass spectrometry and electron energy-loss spectroscopy. We show that the metal-binding site, which appears to be a widespread property of archaellin, is required for filament integrity.
History
DepositionDec 26, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 13, 2019Provider: repository / Type: Initial release
Revision 1.1May 1, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 15, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_abbrev / _citation.journal_volume
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Flagellin
B: Flagellin
C: Flagellin
D: Flagellin
E: Flagellin
F: Flagellin
G: Flagellin
H: Flagellin
I: Flagellin
J: Flagellin
K: Flagellin
L: Flagellin
M: Flagellin
N: Flagellin
O: Flagellin
P: Flagellin
Q: Flagellin
R: Flagellin


Theoretical massNumber of molelcules
Total (without water)389,50818
Polymers389,50818
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: scanning transmission electron microscopy, helical diffraction
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area66190 Å2
ΔGint-503 kcal/mol
Surface area151850 Å2

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Components

#1: Protein
Flagellin


Mass: 21639.359 Da / Num. of mol.: 18 / Source method: isolated from a natural source
Source: (natural) Methanococcus maripaludis (strain S2 / LL) (archaea)
Strain: S2 / LL / References: UniProt: Q6LWP3

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: M.maripaludis archaellin FlaB1 filament / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Methanococcus maripaludis S2 (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Plasmid: pET15b-H3.1, pET15b-H4, pET15b-H2A, pET15b-H2B, pGEM-T-Easy-(186 base-pair mouse ALB1 enhancer DNA)
Buffer solutionpH: 7
Buffer componentName: Water / Formula: H2O
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: sample was monodisperse
Specimen supportDetails: Gatan Solarus / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K / Details: 3 second blot, 3.5uL

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: nanoprobe, parallel beam illumination
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 47619 X / Nominal defocus max: -2500 nm / Nominal defocus min: -1500 nm / Calibrated defocus min: -1500 nm / Calibrated defocus max: -2500 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): 100 K / Temperature (min): 77 K / Residual tilt: 0.1 mradians
Image recordingAverage exposure time: 12 sec. / Electron dose: 96 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2000
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 4 / Used frames/image: 1-48

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1EMAN22.1particle selectione2helixboxer.py
2Leginon3.2image acquisition
4CTFFIND4.1CTF correction
7Cootmodel fitting
9SPRING0.84initial Euler assignment
10SPRING0.84final Euler assignment
11SPRING0.84classificationsparx k-means
12SPRING0.843D reconstruction
13PHENIXmodel refinement
Image processingDetails: frame alignment and integration with motioncor2 incl. dose weighting and 2x Fourier cropping
CTF correctionDetails: deconvolution in SPRING / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 108.2 ° / Axial rise/subunit: 5.7 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 110747
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110747 / Algorithm: FOURIER SPACE / Num. of class averages: 100 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00425848
ELECTRON MICROSCOPYf_angle_d0.96935244
ELECTRON MICROSCOPYf_dihedral_angle_d4.35815318
ELECTRON MICROSCOPYf_chiral_restr0.0694464
ELECTRON MICROSCOPYf_plane_restr0.0064554

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