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- EMDB-20114: CryoEM structure of PilB from Geobacter metallireducens: C2ccocco... -

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Basic information

Entry
Database: EMDB / ID: EMD-20114
TitleCryoEM structure of PilB from Geobacter metallireducens: C2ccocco conformation
Map dataSharpened and z-flipped map
Sample
  • Complex: PilB Hexamer
    • Protein or peptide: Type IV pilus biogenesis ATPase PilB
KeywordsATPase / T4P / type iv pilus / motor / MOTOR PROTEIN
Function / homology
Function and homology information


pilus assembly / ATP hydrolysis activity / ATP binding / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
ATPase, type IV, pilus assembly, PilB / Type II secretion system protein GspE, N-terminal / MshEN domain / Type II secretion system protein GspE, N-terminal superfamily / Bacterial type II secretion system protein E signature. / Type II/IV secretion system protein / Type II/IV secretion system protein / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Type IV pilus biogenesis ATPase PilB
Similarity search - Component
Biological speciesGeobacter metallireducens (bacteria) / Geobacter metallireducens (strain GS-15 / ATCC 53774 / DSM 7210) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsMcCallum M / Howell PL
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nat Commun / Year: 2019
Title: Multiple conformations facilitate PilT function in the type IV pilus.
Authors: Matthew McCallum / Samir Benlekbir / Sheryl Nguyen / Stephanie Tammam / John L Rubinstein / Lori L Burrows / P Lynne Howell /
Abstract: Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by ...Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C symmetry; however, most of these ATPases crystallize with either C or C symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C C, and C conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.
History
DepositionApr 16, 2019-
Header (metadata) releaseJun 5, 2019-
Map releaseNov 20, 2019-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.281
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.281
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6olj
  • Surface level: 0.281
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20114.png

Downloads & links

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Map

FileDownload / File: emd_20114.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened and z-flipped map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.45 Å/pix.
x 256 pix.
= 371.2 Å		1.45 Å/pix.
x 256 pix.
= 371.2 Å		1.45 Å/pix.
x 256 pix.
= 371.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.45 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.281 / Movie #1: 0.281
Minimum - Maximum-0.6185528 - 1.2221209
Average (Standard dev.)-0.001202751 (±0.047119264)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 371.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.451.451.45
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z371.200371.200371.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.6191.222-0.001

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Supplemental data

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Additional map: Original unsharpened and unflipped map

Fileemd_20114_additional.map
AnnotationOriginal unsharpened and unflipped map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PilB Hexamer

EntireName: PilB Hexamer
Components
  • Complex: PilB Hexamer
    • Protein or peptide: Type IV pilus biogenesis ATPase PilB

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Supramolecule #1: PilB Hexamer

SupramoleculeName: PilB Hexamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Geobacter metallireducens (bacteria)

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Macromolecule #1: Type IV pilus biogenesis ATPase PilB

MacromoleculeName: Type IV pilus biogenesis ATPase PilB / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Geobacter metallireducens (strain GS-15 / ATCC 53774 / DSM 7210) (bacteria)
Strain: GS-15 / ATCC 53774 / DSM 7210
Molecular weightTheoretical: 64.899281 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MQASRLGELL VRNNVITKEQ LAKALEEQKS ADGQQRLGSI LIKNGLISEP DLTSFLSKQY GVPSINLSE FEAEQAVVKI IPADVAQKYQ IVPVNRAGST LIIAMADPSN IFAIDDIKFM TGYNVEVVVA SESAIKAAID K YYDQSASL ...String:
MGSSHHHHHH SSGLVPRGSH MQASRLGELL VRNNVITKEQ LAKALEEQKS ADGQQRLGSI LIKNGLISEP DLTSFLSKQY GVPSINLSE FEAEQAVVKI IPADVAQKYQ IVPVNRAGST LIIAMADPSN IFAIDDIKFM TGYNVEVVVA SESAIKAAID K YYDQSASL ADVMGDLEMD DLEVIDTDDE VDVSSLERAT EDAPVVKLVN LILTDAIKRK ASDIHIEPYE RSFRVRYRID GV LYEVMKP PLKLKNAITS RIKIMAELDI AERRLPQDGR IKIKLGGGQD MDYRVSVLPT LFGEKVVLRL LDKSNLQLDM TKL GYEPDA LHYFKEAIHK PFGMVLVTGP TGSGKTVSLY SALGELNKTT ENISTAEDPV EFNFAGINQV QMHEDIGLNF AAAL RSFLR QDPDIIMIGE IRDFETAEIA IKAALTGHLV LSTLHTNDAP ATINRLLNMG VEPFLVASAV NLITAQRLAR RVCSE CKQP EEIPIQALID AGVSPDEAPS YVCYKGTGCV KCNNTGYKGR VGFYQVMPML EEIRELILNG ANTAEIKRES MRLGIK TMR QSGLTKLKEG VTSFEEVLRV TVADD

UniProtKB: Type IV pilus biogenesis ATPase PilB

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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

Concentration0.6 mg/mL
BufferpH: 8
GridModel: Homemade / Material: GOLD
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TECNAI 20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 35.7 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5tsg

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 3257
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5tsg

DetailsInitial fitting in chimera, flexible fitting in Phenix-refine
RefinementProtocol: FLEXIBLE FIT / Overall B value: 68.8
Output model

PDB-6olj:
CryoEM structure of PilB from Geobacter metallireducens: C2ccocco conformation

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