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- PDB-7sb2: Structure of the periplasmic domain of GldM from Capnocytophaga c... -

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Basic information

Entry
Database: PDB / ID: 7sb2
TitleStructure of the periplasmic domain of GldM from Capnocytophaga canimorsus
ComponentsGldM
KeywordsMOTOR PROTEIN / type IX secretion system
Function / homologyGliding motility-associated protein GldM / Gliding motility-associated protein GldM, C-terminal / Gliding motility-associated protein GldM, N-terminal / GldM C-terminal domain / GldM N-terminal domain / Gliding motility protein GldM
Function and homology information
Biological speciesCapnocytophaga canimorsus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHennell James, R. / Deme, J.C. / Lea, S.M.
Funding supportEuropean Union, 4items
OrganizationGrant numberCountry
Wellcome Trust102164/Z/13/ZEuropean Union
Wellcome Trust107929/Z/15/ZEuropean Union
Wellcome Trust219477/Z/19/ZEuropean Union
European Research Council (ERC)833713European Union
CitationJournal: mBio / Year: 2022
Title: Structures of the Type IX Secretion/Gliding Motility Motor from across the Phylum .
Authors: Rory Hennell James / Justin C Deme / Alicia Hunter / Ben C Berks / Susan M Lea /
Abstract: Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are ...Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. Many bacteria in the phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.
History
DepositionSep 23, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 23, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jul 20, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GldM
B: GldM


Theoretical massNumber of molelcules
Total (without water)82,2482
Polymers82,2482
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: homology, Homologous structures have the same stoichiometry.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8030 Å2
ΔGint-58 kcal/mol
Surface area26200 Å2

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Components

#1: Protein GldM


Mass: 41123.895 Da / Num. of mol.: 2 / Fragment: C-terminal TEV cleavage site and TwinStrep Tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Capnocytophaga canimorsus (strain 5) (bacteria)
Gene: Ccan_01630 / Plasmid: pT12 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: F9YQB7
Sequence detailsC-terminal TEV cleavage site and TwinStrep Tag

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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: Type IX Secretion System/gliding motility GldM periplasmic domain
Type: COMPLEX
Details: The structure of the periplasmic domain of GldM was solved using a sample that also included GldL and was used to solve a structure of GldLM
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Capnocytophaga canimorsus Cc5 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 / Plasmid: pT12
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMTris-HClTrisNH2C(CH2OH)3.HCl1
2150 mMsodium chlorideNaClSodium chloride1
30.01 %Lauryl Maltose Neopentyl GlycolC47H88O221
41 mMEthylenediaminetetraacetic acidC10H16N2O81
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The structure of the periplasmic domain of GldM was solved using a sample that also included GldL and was used to solve a structure of GldLM
Specimen supportDetails: 15 mA / Grid material: GOLD / 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: 277.15 K / Details: Wait time 10 s Blot time 2 s

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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
Image recordingElectron dose: 59 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4SIMPLE3CTF correction
7Coot0.9.4.1model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9197926
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 595559 / Symmetry type: POINT
Atomic model buildingB value: 93.02 / Protocol: AB INITIO MODEL / Space: REAL
Details: A homology model based on the structure of FjoGldM (PDB 6EY4) was used as a starting model and modified to fit the EM density using Coot. Refinement was carried out using Phenix.

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