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- PDB-6vk9: Cryo-EM structure of PilA-N/C from Geobacter sulfurreducens -

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Basic information

Entry
Database: PDB / ID: 6vk9
TitleCryo-EM structure of PilA-N/C from Geobacter sulfurreducens
Components
  • Geopilin domain 1 protein
  • Geopilin domain 2 protein
KeywordsPROTEIN FIBRIL / Protein transport / Pili
Function / homology
Function and homology information


pilus assembly / protein secretion by the type II secretion system / type II protein secretion system complex / membrane
Similarity search - Function
Bacterial general secretion pathway protein G-type pilin / Prokaryotic N-terminal methylation site. / Prokaryotic N-terminal methylation motif / Prokaryotic N-terminal methylation site / Pilin-like
Similarity search - Domain/homology
Geopilin domain 2 protein / Geopilin domain 1 protein
Similarity search - Component
Biological speciesGeobacter sulfurreducens (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsGu, Y. / Srikanth, V. / Malvankar, N.S. / Samatey, F.A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1749662 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2AI138259-01 United States
Department of Defense (DOD, United States)W911NF-18-2-0100 United States
National Institutes of Health/Office of the DirectorNew Innovator Award United States
CitationJournal: Nature / Year: 2021
Title: Structure of Geobacter pili reveals secretory rather than nanowire behaviour.
Authors: Yangqi Gu / Vishok Srikanth / Aldo I Salazar-Morales / Ruchi Jain / J Patrick O'Brien / Sophia M Yi / Rajesh Kumar Soni / Fadel A Samatey / Sibel Ebru Yalcin / Nikhil S Malvankar /
Abstract: Extracellular electron transfer by Geobacter species through surface appendages known as microbial nanowires is important in a range of globally important environmental phenomena, as well as for ...Extracellular electron transfer by Geobacter species through surface appendages known as microbial nanowires is important in a range of globally important environmental phenomena, as well as for applications in bio-remediation, bioenergy, biofuels and bioelectronics. Since 2005, these nanowires have been thought to be type 4 pili composed solely of the PilA-N protein. However, previous structural analyses have demonstrated that, during extracellular electron transfer, cells do not produce pili but rather nanowires made up of the cytochromes OmcS and OmcZ. Here we show that Geobacter sulfurreducens binds PilA-N to PilA-C to assemble heterodimeric pili, which remain periplasmic under nanowire-producing conditions that require extracellular electron transfer. Cryo-electron microscopy revealed that C-terminal residues of PilA-N stabilize its copolymerization with PilA-C (to form PilA-N-C) through electrostatic and hydrophobic interactions that position PilA-C along the outer surface of the filament. PilA-N-C filaments lack π-stacking of aromatic side chains and show a conductivity that is 20,000-fold lower than that of OmcZ nanowires. In contrast with surface-displayed type 4 pili, PilA-N-C filaments show structure, function and localization akin to those of type 2 secretion pseudopili. The secretion of OmcS and OmcZ nanowires is lost when pilA-N is deleted and restored when PilA-N-C filaments are reconstituted. The substitution of pilA-N with the type 4 pili of other microorganisms also causes a loss of secretion of OmcZ nanowires. As all major phyla of prokaryotes use systems similar to type 4 pili, this nanowire translocation machinery may have a widespread effect in identifying the evolution and prevalence of diverse electron-transferring microorganisms and in determining nanowire assembly architecture for designing synthetic protein nanowires.
History
DepositionJan 19, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 7, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 1, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.pdbx_database_id_DOI / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Sep 29, 2021Group: Database references / Category: citation
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.title / _citation.year
Revision 1.3May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: Geopilin domain 1 protein
O: Geopilin domain 1 protein
Q: Geopilin domain 1 protein
S: Geopilin domain 1 protein
U: Geopilin domain 1 protein
G: Geopilin domain 1 protein
I: Geopilin domain 1 protein
K: Geopilin domain 1 protein
M: Geopilin domain 1 protein
C: Geopilin domain 1 protein
E: Geopilin domain 1 protein
W: Geopilin domain 1 protein
Y: Geopilin domain 1 protein
0: Geopilin domain 1 protein
2: Geopilin domain 1 protein
4: Geopilin domain 1 protein
D: Geopilin domain 2 protein
B: Geopilin domain 2 protein
P: Geopilin domain 2 protein
R: Geopilin domain 2 protein
T: Geopilin domain 2 protein
V: Geopilin domain 2 protein
H: Geopilin domain 2 protein
J: Geopilin domain 2 protein
L: Geopilin domain 2 protein
N: Geopilin domain 2 protein
F: Geopilin domain 2 protein
X: Geopilin domain 2 protein
Z: Geopilin domain 2 protein
1: Geopilin domain 2 protein
3: Geopilin domain 2 protein
5: Geopilin domain 2 protein


Theoretical massNumber of molelcules
Total (without water)280,80432
Polymers280,80432
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 16 / Rise per n subunits: 10.41 Å / Rotation per n subunits: 89.03 °)

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Components

#1: Protein
Geopilin domain 1 protein


Mass: 6576.502 Da / Num. of mol.: 16 / Fragment: UNP residues 30-90
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Gene: pilA-N, GSU1496 / Production host: Geobacter sulfurreducens (bacteria) / References: UniProt: Q74D23
#2: Protein
Geopilin domain 2 protein


Mass: 10973.763 Da / Num. of mol.: 16 / Fragment: UNP residues 21-124
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Gene: pilA-C, GSU1497 / Production host: Geobacter sulfurreducens (bacteria) / References: UniProt: Q74D22

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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: Polymerized hetero-dimers of PilA-N and PilA-C / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
10.0075 mMDisodium phosphateNa2HPO41
20.0025 mMMonosodium phosphateNaH2PO41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: 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 FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 2.308 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: 7664
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 27 / Used frames/image: 2-10

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Processing

EM software
IDNameVersionCategory
1RELION3.05particle selection
2SerialEMimage acquisition
4Gctf1.08CTF correction
7UCSF Chimera1.13model fitting
9RELION3.05initial Euler assignment
10RELION3.05final Euler assignment
12RELION3.053D reconstruction
13PHENIXdev_3689model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 89.01 ° / Axial rise/subunit: 10.41 Å / Axial symmetry: C1
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83000 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
RefinementHighest resolution: 3.8 Å

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