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- EMDB-0089: Cryo-EM structure of the bacteria-killing type IV secretion syste... -

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Basic information

Entry
Database: EMDB / ID: EMD-0089
TitleCryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citriSecretion
Map data
Sample
  • Complex: Core complex of a bacterial killing type IV secretion system from XanthomonasSecretion
    • Protein or peptide: VirB7
    • Protein or peptide: VirB9 protein
    • Protein or peptide: VirB10 protein
Function / homology
Function and homology information


membrane => GO:0016020
Similarity search - Function
Toxin co-regulated pilus biosynthesis protein Q, C-terminal / Toxin co-regulated pilus biosynthesis protein Q / Conjugal transfer, TrbG/VirB9/CagX / VirB9/CagX/TrbG, C-terminal / VirB9/CagX/TrbG, C-terminal domain superfamily / Conjugal transfer protein / Type IV secretion system, VirB10/TrbI / Bacterial conjugation TrbI-like protein / Type IV secretion system, VirB10 / TraB / TrbI
Similarity search - Domain/homology
Toxin co-regulated pilus biosynthesis protein Q C-terminal domain-containing protein / VirB9 protein / VirB10 protein
Similarity search - Component
Biological speciesXanthomonas axonopodis pv. citri str. 306 (bacteria) / Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsCosta TRD / Sgro GG / Farah CS / Waksman G
Funding support United Kingdom, Brazil, 3 items
OrganizationGrant numberCountry
Wellcome Trust098302 United Kingdom
Sao Paulo Research Foundation2017/17303-7 Brazil
Sao Paulo Research Foundation2011/07777-5 Brazil
CitationJournal: Nat Microbiol / Year: 2018
Title: Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri.
Authors: Germán G Sgro / Tiago R D Costa / William Cenens / Diorge P Souza / Alexandre Cassago / Luciana Coutinho de Oliveira / Roberto K Salinas / Rodrigo V Portugal / Chuck S Farah / Gabriel Waksman /
Abstract: Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically ...Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein-protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution.
History
DepositionJun 28, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseOct 24, 2018-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6gyb
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0089.png

Downloads & links

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Map

FileDownload / File: emd_0089.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.16240412 - 0.27459824
Average (Standard dev.)0.0006112758 (±0.008838454)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 370.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z371.000371.000371.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.1620.2750.001

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Supplemental data

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Sample components

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Entire : Core complex of a bacterial killing type IV secretion system from...

EntireName: Core complex of a bacterial killing type IV secretion system from XanthomonasSecretion
Components
  • Complex: Core complex of a bacterial killing type IV secretion system from XanthomonasSecretion
    • Protein or peptide: VirB7
    • Protein or peptide: VirB9 protein
    • Protein or peptide: VirB10 protein

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Supramolecule #1: Core complex of a bacterial killing type IV secretion system from...

SupramoleculeName: Core complex of a bacterial killing type IV secretion system from Xanthomonas
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fourteen copies of each of the following three subunits: VirB7, VirB9 and VirB10
Source (natural)Organism: Xanthomonas axonopodis pv. citri str. 306 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: VirB7

MacromoleculeName: VirB7 / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Molecular weightTheoretical: 14.762795 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MNPMYVSKLS LVLVAAALVG ACATKPAPDF GGRWKHVNHF DEAPTEIPLY TSYTYQATPM DGTLKTMLER WAADSNMQLS YNLPSDYTL IGPVSAISTT SVQQAATELS AVYAAQGVSV SVSANKLLVQ PVPVSSGAKL

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Macromolecule #2: VirB9 protein

MacromoleculeName: VirB9 protein / type: protein_or_peptide / ID: 2 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Strain: 306
Molecular weightTheoretical: 29.359385 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKLFNRYRVA LLSALPLALC ALSAAAQVVQ EYEYAPDRIY QVRTGLGITT QVELSPNEKI LDYSTGFTGG WELTRRENVF YLKPKNVDV DTNMMIRTAT HSYILELKVV ATDWQRLEQA KQAGVQYKVV FTYPKDTSFN NVADADTSKN GPLLNAKILK D RRYYYDYD ...String:
MKLFNRYRVA LLSALPLALC ALSAAAQVVQ EYEYAPDRIY QVRTGLGITT QVELSPNEKI LDYSTGFTGG WELTRRENVF YLKPKNVDV DTNMMIRTAT HSYILELKVV ATDWQRLEQA KQAGVQYKVV FTYPKDTSFN NVADADTSKN GPLLNAKILK D RRYYYDYD YATRTKKSWL IPSRVYDDGK FTYINMDLTR FPTGNFPAVF AREKEHAEDF LVNTTVEGNT LIVHGTYPFL VV RHGDNVV GLRRNKQK

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Macromolecule #3: VirB10 protein

MacromoleculeName: VirB10 protein / type: protein_or_peptide / ID: 3 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Strain: 306
Molecular weightTheoretical: 43.392469 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNSNIPNSPD ERIQNHGGDE QHNGDHNERN NPYFARQQAS AEPDLDANEP ILRSSDIKRL NRKALVFLAA IAALLILAIF WLATQSGED SAPPKPRTET VVAPALPQSM TAPVEEAPVP LAQQPSLPPL PPMPTDNSEE VSSAPERQRG PTLLERRILA E SAANGGGV ...String:
MNSNIPNSPD ERIQNHGGDE QHNGDHNERN NPYFARQQAS AEPDLDANEP ILRSSDIKRL NRKALVFLAA IAALLILAIF WLATQSGED SAPPKPRTET VVAPALPQSM TAPVEEAPVP LAQQPSLPPL PPMPTDNSEE VSSAPERQRG PTLLERRILA E SAANGGGV PGQLGAQPAP TQEDGPVTLA KPISNPDGLL VRGTYIRCIL ETRIISDFGG YTSCIVTEPV YSINGHNLLL PK GSKMLGQ YSAGEPTSHR LQVVWDRVTT PTGLDVTLMG PGIDTLGSSG HPGNYNAHWG NKIASALFIS LLSDAFKYAA AEY GPETTT IGVGSGIVTQ QPFESNTARS MQQLAEQAVE KSGRRPATLT INQGTVLNVY VAKDVDFSAV LPKAAALEGL SAWS HPQFE K

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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

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMNH2C(CH2OH)3HClTris-HClTris
200.0 mMNaClSodium chlorideSodium Chloride
10.0 mMCH3(CH2)11N(O)(CH3)2LDAO
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.2 K / Instrument: FEI VITROBOT MARK IV
Details: Blot for 4.5 seconds after 30 seconds of incubation..

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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 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 1469 / Average exposure time: 12.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 185079
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1.0)
Final 3D classificationNumber classes: 1 / Avg.num./class: 142306 / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C14 (14 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 142306
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe electron density was clearly interpretable, which allowed us to build a de novo structural model. This process began by fitting the crystallographic model of the X. citri VirB7 C-terminal N0 domain (PDB:3OV5) and the NMR model of the X. citri VirB9CTD-VirB7NTD complex (PDB:2N01) in order to identify the map with the correct handedness. Models were positioned using Fit in map tool in Chimera, and saved relative to the map. Using these as starting points, we were able to manually build the rest of the model for VirB7 and VirB9CTD, and the de novo models for VirB10CTD, VirB10NTD_150-161 and VirB9NTD using Coot. In this manner, we obtained a combined model for a single VirB7-VirB9-VirB10 heterotrimer unit, which was submitted to iterative rounds of real space refinement and building using PHENIX and Coot software, respectively. Thirteen more copies of the refined heterotrimer were then fit into the density map using Chimera and new rounds of real space refinement (now using NCS for the 42 chains contained in the structure) and building using PHENIX and Coot, respectively, were executed until we obtained good parameters for Ramachandran plot and MolProbity. Chimera and PyMol were used for map and model visualization and figure production.
RefinementSpace: REAL / Overall B value: 138 / Target criteria: Cross-correlation coefficient
Output model

PDB-6gyb:
Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri

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