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- PDB-8gqy: CryoEM structure of pentameric MotA from Aquifex aeolicus -

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Basic information

Entry
Database: PDB / ID: 8gqy
TitleCryoEM structure of pentameric MotA from Aquifex aeolicus
ComponentsMotility protein A
KeywordsMEMBRANE PROTEIN / Bacterial flagellum / stator protein / Aquifex aeolicus / single particle Cryo-EM / MotA
Function / homology
Function and homology information


bacterial-type flagellum-dependent swarming motility / proton transmembrane transport / chemotaxis / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Flagellar motor protein MotA, conserved site / Flagellar motor protein motA family signature. / MotA/TolQ/ExbB proton channel / MotA/TolQ/ExbB proton channel family
Similarity search - Domain/homology
Biological speciesAquifex aeolicus VF5 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsNishikino, T. / Takekawa, N. / Kishikawa, J. / Hirose, M. / Onoe, S. / Kato, T. / Imada, K.
Funding support Japan, 7items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP20J00329 Japan
Japan Society for the Promotion of Science (JSPS)JP16J01859 Japan
Japan Society for the Promotion of Science (JSPS)JP20K15732 Japan
Japan Society for the Promotion of Science (JSPS)JP20K06514 Japan
Japan Society for the Promotion of Science (JSPS)JP22K18359 Japan
Japan Society for the Promotion of Science (JSPS)JP22H02559 Japan
Japan Society for the Promotion of Science (JSPS)JP21H02443 Japan
CitationJournal: Biochem Biophys Res Commun / Year: 2022
Title: Structure of MotA, a flagellar stator protein, from hyperthermophile.
Authors: Tatsuro Nishikino / Norihiro Takekawa / Duy Phuoc Tran / Jun-Ichi Kishikawa / Mika Hirose / Sakura Onoe / Seiji Kojima / Michio Homma / Akio Kitao / Takayuki Kato / Katsumi Imada /
Abstract: Many motile bacteria swim and swarm toward favorable environments using the flagellum, which is rotated by a motor embedded in the inner membrane. The motor is composed of the rotor and the stator, ...Many motile bacteria swim and swarm toward favorable environments using the flagellum, which is rotated by a motor embedded in the inner membrane. The motor is composed of the rotor and the stator, and the motor torque is generated by the change of the interaction between the rotor and the stator induced by the ion flow through the stator. A stator unit consists of two types of membrane proteins termed A and B. Recent cryo-EM studies on the stators from mesophiles revealed that the stator consists of five A and two B subunits, whereas the low-resolution EM analysis showed that purified hyperthermophilic MotA forms a tetramer. To clarify the assembly formation and factors enhancing thermostability of the hyperthermophilic stator, we determined the cryo-EM structure of MotA from Aquifex aeolicus (Aa-MotA), a hyperthermophilic bacterium, at 3.42 Å resolution. Aa-MotA forms a pentamer with pseudo C5 symmetry. A simulated model of the Aa-MotAMotB stator complex resembles the structures of mesophilic stator complexes, suggesting that Aa-MotA can assemble into a pentamer equivalent to the stator complex without MotB. The distribution of hydrophobic residues of MotA pentamers suggests that the extremely hydrophobic nature in the subunit boundary and the transmembrane region is a key factor to stabilize hyperthermophilic Aa-MotA.
History
DepositionAug 31, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 26, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2022Group: Data collection / Source and taxonomy / Structure summary
Category: em_software / entity_src_gen / pdbx_contact_author
Item: _entity_src_gen.pdbx_gene_src_scientific_name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Motility protein A
B: Motility protein A
C: Motility protein A
D: Motility protein A
E: Motility protein A


Theoretical massNumber of molelcules
Total (without water)144,3165
Polymers144,3165
Non-polymers00
Water0
1


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

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Components

#1: Protein
Motility protein A / / Chemotaxis protein MotA


Mass: 28863.195 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Details: Met1 to His6 is translation enhancing element sequence. 6 His residues on C-terminal are purification tag.
Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Strain: VF5 / Gene: motA, aq_1003 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: O67122

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Pentameric MotA from Aquifex aeolicus / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: NO
Source (natural)Organism: Aquifex aeolicus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMTris-HClTrisC4H11NO31
30.01 % (w/v)LMNGC47H88O221
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: MOLYBDENUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 60 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.22 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 6299
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2240219
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 482752 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6YKM

6ykm

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059655
ELECTRON MICROSCOPYf_angle_d0.55813075
ELECTRON MICROSCOPYf_dihedral_angle_d13.375890
ELECTRON MICROSCOPYf_chiral_restr0.041610
ELECTRON MICROSCOPYf_plane_restr0.0041630

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