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- PDB-6sm3: Structure of the RagAB peptide importer in the 'closed-closed' state -

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Basic information

Entry
Database: PDB / ID: 6sm3
TitleStructure of the RagAB peptide importer in the 'closed-closed' state
Components
  • GLY-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-THR-SER
  • Lipoprotein RagB
  • RagA protein
KeywordsMEMBRANE PROTEIN / Beta-barrel / OMP / TonB-dependent / transporter
Function / homology
Function and homology information


cell outer membrane / membrane
Similarity search - Function
TonB-dependent outer membrane protein, SusC/RagA / TonB-dependent outer membrane protein SusC/RagA, conserved site / CarboxypepD_reg-like domain / SusD-like, N-terminal / Starch-binding associating with outer membrane / RagB/SusD domain / SusD family / Carboxypeptidase-like, regulatory domain superfamily / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor ...TonB-dependent outer membrane protein, SusC/RagA / TonB-dependent outer membrane protein SusC/RagA, conserved site / CarboxypepD_reg-like domain / SusD-like, N-terminal / Starch-binding associating with outer membrane / RagB/SusD domain / SusD family / Carboxypeptidase-like, regulatory domain superfamily / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor / TonB-dependent receptor, plug domain superfamily / TonB-dependent receptor, plug domain / TonB-dependent receptor-like, beta-barrel domain superfamily / TonB-dependent Receptor Plug Domain / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
Chem-5PL / PALMITIC ACID / Lipoprotein RagB / RagA protein
Similarity search - Component
Biological speciesPorphyromonas gingivalis (bacteria)
Porphyromonas gingivalis W83 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWhite, J.B.R. / Ranson, N.A. / van den Berg, B.
Funding support United Kingdom, Poland, 7items
OrganizationGrant numberCountry
Wellcome Trust214222/Z/18/Z United Kingdom
Wellcome Trust215064/Z/18/Z United Kingdom
Polish National Science CentreUMO-2015/19/N/NZ1/00322 Poland
Polish National Science CentreUMO-2018/28/T/NZ1/00348 to MM Poland
Polish National Science CentreUMO-2016/23/N/NZ1/01513 Poland
Polish National Science CentreUMO-2018/31/B/NZ1/03968 Poland
Polish National Science CentreNIDCR/DE 022597 Poland
CitationJournal: Nat Microbiol / Year: 2020
Title: Structural and functional insights into oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis.
Authors: Mariusz Madej / Joshua B R White / Zuzanna Nowakowska / Shaun Rawson / Carsten Scavenius / Jan J Enghild / Grzegorz P Bereta / Karunakar Pothula / Ulrich Kleinekathoefer / Arnaud Baslé / ...Authors: Mariusz Madej / Joshua B R White / Zuzanna Nowakowska / Shaun Rawson / Carsten Scavenius / Jan J Enghild / Grzegorz P Bereta / Karunakar Pothula / Ulrich Kleinekathoefer / Arnaud Baslé / Neil A Ranson / Jan Potempa / Bert van den Berg /
Abstract: Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory ...Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagAB complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a 'pedal bin' mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis.
History
DepositionAug 21, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 20, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Derived calculations / Category: citation / citation_author / struct_conn
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

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Assembly

Deposited unit
A: Lipoprotein RagB
B: RagA protein
C: GLY-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-THR-SER
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,4285
Polymers155,9383
Non-polymers1,4902
Water0
1
A: Lipoprotein RagB
B: RagA protein
C: GLY-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-THR-SER
hetero molecules

A: Lipoprotein RagB
B: RagA protein
C: GLY-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-THR-SER
hetero molecules


Theoretical massNumber of molelcules
Total (without water)314,85610
Polymers311,8766
Non-polymers2,9804
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation1
Buried area9660 Å2
ΔGint-45 kcal/mol
Surface area57140 Å2
MethodPISA

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Components

#1: Protein Lipoprotein RagB


Mass: 54430.863 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Porphyromonas gingivalis (strain ATCC BAA-308 / W83) (bacteria)
Strain: ATCC BAA-308 / W83 / References: UniProt: F5H948
#2: Protein RagA protein /


Mass: 100350.977 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Porphyromonas gingivalis (strain ATCC BAA-308 / W83) (bacteria)
Strain: ATCC BAA-308 / W83 / References: UniProt: Q7MXJ7
#3: Protein/peptide GLY-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-THR-SER


Mass: 1156.157 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Putative peptide substrate / Source: (natural) Porphyromonas gingivalis W83 (bacteria) / Strain: KRAB
#4: Chemical ChemComp-5PL / (1R,4S,6R)-6-({[2-(ACETYLAMINO)-2-DEOXY-ALPHA-D-GLUCOPYRANOSYL]OXY}METHYL)-4-HYDROXY-1-{[(15-METHYLHEXADECANOYL)OXY]METHYL}-4-OXIDO-7-OXO-3,5-DIOXA-8-AZA-4-PHOSPHAHEPTACOS-1-YL 15-METHYLHEXADECANOATE


Mass: 1233.719 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C67H129N2O15P
#5: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2
Has ligand of interestN

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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: RagAB with putative peptide substrate / Type: COMPLEX
Details: Putative peptide substrate co-purified with the complex
Entity ID: #1-#3 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Porphyromonas gingivalis W83 (bacteria) / Strain: KRAB
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
2100 mMSodium chlorideNaClSodium chloride1
30.03 % w/vDDMC24H46O111
SpecimenConc.: 1.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / 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: 279.15 K / Details: 6 second blot time, blot force 6

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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: 77.88 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: (1.14_3260: phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1particle selection
2EPUimage acquisition
4GctfCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86877 / Symmetry type: POINT

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