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- EMDB-10245: Structure of the RagAB peptide importer in the 'open-closed' state -

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Basic information

Entry
Database: EMDB / ID: EMD-10245
TitleStructure of the RagAB peptide importer in the 'open-closed' state
Map dataSharpened map filtered by local resolution in RELION
Sample
  • Complex: RagAB with putative peptide substrate
    • Protein or peptide: Lipoprotein RagB
    • Protein or peptide: RagA protein
    • Protein or peptide: SER-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-ASN-SER
    • Protein or peptide: RagA protein
  • Ligand: (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
  • Ligand: PALMITIC ACID
  • Ligand: (HYDROXYETHYLOXY)TRI(ETHYLOXY)OCTANE
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
Lipoprotein RagB / RagA protein
Similarity search - Component
Biological speciesPorphyromonas gingivalis W83 (bacteria) / Porphyromonas gingivalis (strain ATCC BAA-308 / W83) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWhite JBR / Ranson NA / van den Berg B
Funding support United Kingdom, Poland, 7 items
OrganizationGrant numberCountry
Wellcome Trust214222/Z/18/Z United Kingdom
Polish National Science CentreUMO-2015/19/N/NZ1/00322 Poland
Wellcome Trust215064/Z/18/Z United Kingdom
Polish National Science CentreUMO-2016/23/N/NZ1/01513 Poland
Polish National Science CentreUMO-2018/28/T/NZ1/00348 to MM Poland
Polish National Science CentreNIDCR/DE 022597 Poland
Polish National Science CentreUMO-2018/31/B/NZ1/03968 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 22, 2019-
Header (metadata) releaseSep 4, 2019-
Map releaseMay 20, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6smq
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10245.png

Downloads & links

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Map

FileDownload / File: emd_10245.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map filtered by local resolution in RELION
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.3826556 - 0.5361876
Average (Standard dev.)0.0005051065 (±0.019097699)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 231.12001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z216216216
origin x/y/z0.0000.0000.000
length x/y/z231.120231.120231.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS216216216
D min/max/mean-0.3830.5360.001

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Supplemental data

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Mask #1

Fileemd_10245_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map from 3D auto-refinement in RELION

Fileemd_10245_additional_1.map
AnnotationUnsharpened map from 3D auto-refinement in RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half map 1 from final iteration of 3D auto-refinement in RELION

Fileemd_10245_additional_2.map
AnnotationHalf map 1 from final iteration of 3D auto-refinement in RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half map 2 from final iteration of 3D auto-refinement in RELION

Fileemd_10245_additional_3.map
AnnotationHalf map 2 from final iteration of 3D auto-refinement in RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RagAB with putative peptide substrate

EntireName: RagAB with putative peptide substrate
Components
  • Complex: RagAB with putative peptide substrate
    • Protein or peptide: Lipoprotein RagB
    • Protein or peptide: RagA protein
    • Protein or peptide: SER-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-ASN-SER
    • Protein or peptide: RagA protein
  • Ligand: (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
  • Ligand: PALMITIC ACID
  • Ligand: (HYDROXYETHYLOXY)TRI(ETHYLOXY)OCTANE

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Supramolecule #1: RagAB with putative peptide substrate

SupramoleculeName: RagAB with putative peptide substrate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Putative peptide substrate co-purified with the complex
Source (natural)Organism: Porphyromonas gingivalis W83 (bacteria) / Strain: KRAB

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Macromolecule #1: Lipoprotein RagB

MacromoleculeName: Lipoprotein RagB / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Porphyromonas gingivalis (strain ATCC BAA-308 / W83) (bacteria)
Strain: ATCC BAA-308 / W83
Molecular weightTheoretical: 54.430863 KDa
SequenceString: CELDRDPEGK DFQQPYTSFV QTKQNRDGLY ALLRNTENPR MHFYQELQSD MYCTTITDGN SLAPFVNWDL GILNDHGRAD EDEVSGIAG YYFVYNRLNQ QANAFVNNTE AALQNQVYKN STEIANAKSF LAEGKVLQAL AIWRLMDRFS FHESVTEVNS G AKDLGVIL ...String:
CELDRDPEGK DFQQPYTSFV QTKQNRDGLY ALLRNTENPR MHFYQELQSD MYCTTITDGN SLAPFVNWDL GILNDHGRAD EDEVSGIAG YYFVYNRLNQ QANAFVNNTE AALQNQVYKN STEIANAKSF LAEGKVLQAL AIWRLMDRFS FHESVTEVNS G AKDLGVIL LKEYNPGYIG PRATKAQCYD YILSRLSEAI EVLPENRESV LYVSRDYAYA LRARIYLALG EYGKAAADAK MV VDKYPLI GAADASEFEN IYRSDANNPE IIFRGFASAT LGSFTATTLN GAAPAGKDIK YNPSAVPFQW VVDLYENEDF RKS VYIAKV VKKDKGYLVN KFLEDKAYRD VQDKPNLKVG ARYFSVAEVY LILVESALQT GDTPTAEKYL KALSKARGAE VSVV NMEAL QAERTRELIG EGSRLRDMVR WSIPNNHDAF ETQPGLEGFA NTTPLKAQAP VGFYAYTWEF PQRDRQTNPQ LIKNW PI

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Macromolecule #2: RagA protein

MacromoleculeName: RagA protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Porphyromonas gingivalis (strain ATCC BAA-308 / W83) (bacteria)
Strain: ATCC BAA-308 / W83
Molecular weightTheoretical: 100.350977 KDa
SequenceString: LSTVSGSVAK VSSEKLAEKP VANIMDALQG QVAGMQVMTT SGDPTAVASV EIHGTGSLGA SSAPLYIVDG MQTSLDVVAT MNPNDFESM SVLKDASATS IYGARAANGV VFIQTKKGKM SERGRITFNA SYGISQILNT KPLDNMMTGD ELLDFQVKAG F WGNNQTVQ ...String:
LSTVSGSVAK VSSEKLAEKP VANIMDALQG QVAGMQVMTT SGDPTAVASV EIHGTGSLGA SSAPLYIVDG MQTSLDVVAT MNPNDFESM SVLKDASATS IYGARAANGV VFIQTKKGKM SERGRITFNA SYGISQILNT KPLDNMMTGD ELLDFQVKAG F WGNNQTVQ KVKDMILAGA EDLYGNYDSL KDEYGKTLFP VDFNHDADWL KALFKTAPTS QGDISFSGGS QGTSYYASIG YF DQEGMAR EPANFKRYSG RLNFESRINE WLKVGANLSG AIANRRSADY FGKYYMGSGT FGVLTMPRYY NPFDVNGDLA DVY YMYGAT RPSMTEPYFA KMRPFSSESH QANVNGFAQI TPIKGLTLKA QAGVDITNTR TSSKRMPNNP YDSTPLGERR ERAY RDVSK SFTNTAEYKF SIDEKHDLTA LMGHEYIEYE GDVIGASSKG FESDKLMLLS QGKTGNSLSL PEHRVAEYAY LSFFS RFNY GFDKWMYIDF SVRNDQSSRF GSNNRSAWFY SVGGMFDIYN KFIQESNWLS DLRLKMSYGT TGNSEIGNYN HQALVT VNN YTEDAMGLSI STAGNPDLSW EKQSQFNFGL AAGAFNNRLS AEVDFYVRTT NDMLIDVPMP YISGFFSQYQ NVGSMKN TG VDLSLKGTIY QNKDWNVYAS ANFNYNRQEI TKLFFGLNKY MLPNTGTIWE IGYPNSFYMA EYAGIDKKTG KQLWYVPG Q VDADGNKVTT SQYSADLETR IDKSVTPPIT GGFSLGASWK GLSLDADFAY IVGKWMINND RYFTENGGGL MQLNKDKML LNAWTEDNKE TDVPKLGQSP QFDTHLLENA SFLRLKNLKL TYVLPNSLFA GQNVIGGARV YLMARNLLTV TKYKGFDPEA GGNVGKNQY PNSKQYVAGI QLSF

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Macromolecule #3: SER-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-ASN-SER

MacromoleculeName: SER-GLY-ALA-THR-THR-ALA-THR-THR-THR-THR-SER-ASN-SER / type: protein_or_peptide / ID: 3 / Details: Putative peptide ligand / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Porphyromonas gingivalis W83 (bacteria) / Strain: KRAB
Molecular weightTheoretical: 1.199182 KDa
SequenceString:
SGATTATTTT SNS

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Macromolecule #4: RagA protein

MacromoleculeName: RagA protein / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Porphyromonas gingivalis (strain ATCC BAA-308 / W83) (bacteria)
Strain: ATCC BAA-308 / W83
Molecular weightTheoretical: 101.582398 KDa
SequenceString: QVVVLGYGTG QKLSTVSGSV AKVSSEKLAE KPVANIMDAL QGQVAGMQVM TTSGDPTAVA SVEIHGTGSL GASSAPLYIV DGMQTSLDV VATMNPNDFE SMSVLKDASA TSIYGARAAN GVVFIQTKKG KMSERGRITF NASYGISQIL NTKPLDNMMT G DELLDFQV ...String:
QVVVLGYGTG QKLSTVSGSV AKVSSEKLAE KPVANIMDAL QGQVAGMQVM TTSGDPTAVA SVEIHGTGSL GASSAPLYIV DGMQTSLDV VATMNPNDFE SMSVLKDASA TSIYGARAAN GVVFIQTKKG KMSERGRITF NASYGISQIL NTKPLDNMMT G DELLDFQV KAGFWGNNQT VQKVKDMILA GAEDLYGNYD SLKDEYGKTL FPVDFNHDAD WLKALFKTAP TSQGDISFSG GS QGTSYYA SIGYFDQEGM AREPANFKRY SGRLNFESRI NEWLKVGANL SGAIANRRSA DYFGKYYMGS GTFGVLTMPR YYN PFDVNG DLADVYYMYG ATRPSMTEPY FAKMRPFSSE SHQANVNGFA QITPIKGLTL KAQAGVDITN TRTSSKRMPN NPYD STPLG ERRERAYRDV SKSFTNTAEY KFSIDEKHDL TALMGHEYIE YEGDVIGASS KGFESDKLML LSQGKTGNSL SLPEH RVAE YAYLSFFSRF NYGFDKWMYI DFSVRNDQSS RFGSNNRSAW FYSVGGMFDI YNKFIQESNW LSDLRLKMSY GTTGNS EIG NYNHQALVTV NNYTEDAMGL SISTAGNPDL SWEKQSQFNF GLAAGAFNNR LSAEVDFYVR TTNDMLIDVP MPYISGF FS QYQNVGSMKN TGVDLSLKGT IYQNKDWNVY ASANFNYNRQ EITKLFFGLN KYMLPNTGTI WEIGYPNSFY MAEYAGID K KTGKQLWYVP GQVDADGNKV TTSQYSADLE TRIDKSVTPP ITGGFSLGAS WKGLSLDADF AYIVGKWMIN NDRYFTENG GGLMQLNKDK MLLNAWTEDN KETDVPKLGQ SPQFDTHLLE NASFLRLKNL KLTYVLPNSL FAGQNVIGGA RVYLMARNLL TVTKYKGFD PEAGGNVGKN QYPNSKQYVA GIQLSF

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Macromolecule #5: (1R,4S,6R)-6-({[2-(ACETYLAMINO)-2-DEOXY-ALPHA-D-GLUCOPYRANOSYL]OX...

MacromoleculeName: (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
type: ligand / ID: 5 / Number of copies: 2 / Formula: 5PL
Molecular weightTheoretical: 1.233719 KDa
Chemical component information

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

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Macromolecule #6: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 6 / Number of copies: 2 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID / Palmitic acid

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Macromolecule #7: (HYDROXYETHYLOXY)TRI(ETHYLOXY)OCTANE

MacromoleculeName: (HYDROXYETHYLOXY)TRI(ETHYLOXY)OCTANE / type: ligand / ID: 7 / Number of copies: 5 / Formula: C8E
Molecular weightTheoretical: 306.438 Da
Chemical component information

ChemComp-C8E:
(HYDROXYETHYLOXY)TRI(ETHYLOXY)OCTANE / C8E, detergent*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.75 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium chlorideSodium chloride
0.03 % w/vC24H46O11DDM
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279.15 K / Instrument: FEI VITROBOT MARK IV / Details: 6 second blot time, blot force 6.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 77.88 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: Initial model generation in RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 213143
FSC plot (resolution estimation)

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