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- EMDB-20116: CryoEM structure of PilT4 from Geobacter metallireducens without ... -

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Basic information

Entry
Database: EMDB / ID: EMD-20116
TitleCryoEM structure of PilT4 from Geobacter metallireducens without adding nucleotide: C2oocooc conformation
Map dataSharpened and z-flipped map
Sample
  • Complex: PilT Hexamer
    • Protein or peptide: Twitching motility pilus retraction ATPase
KeywordsATPase / T4P / type iv pilus / motor / MOTOR PROTEIN
Function / homologyPilus retraction protein PilT/PilU / 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 / ATP binding / Twitching motility pilus retraction ATPase
Function and homology information
Biological speciesGeobacter metallireducens (bacteria) / Geobacter metallireducens (strain GS-15 / ATCC 53774 / DSM 7210) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 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-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.41
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.41
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6oll
  • Surface level: 0.41
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20116.png

Downloads & links

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Map

FileDownload / File: emd_20116.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened and z-flipped map
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.41 / Movie #1: 0.41
Minimum - Maximum-1.9166515 - 2.2735968
Average (Standard dev.)0.004868001 (±0.07380414)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.360271.360271.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.9172.2740.005

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Supplemental data

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Additional map: Original unsharpened and unflipped map

Fileemd_20116_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 : PilT Hexamer

EntireName: PilT Hexamer
Components
  • Complex: PilT Hexamer
    • Protein or peptide: Twitching motility pilus retraction ATPase

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Supramolecule #1: PilT Hexamer

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

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Macromolecule #1: Twitching motility pilus retraction ATPase

MacromoleculeName: Twitching motility pilus retraction ATPase / 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: 42.883379 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MANMHQLLTE LVNRGGSDLH LTTNSPPQIR IDGKLLPLDM PPLNAVDTKQ LCYSILTEQQ KHKFEENNE LDLSFGIKGL SRFRGNVFVQ RGAVAGVFRV IPYKILSFEE LGLPPVVREL AEKPRGLVLV TGPTGSGKST T LAAIIDKI ...String:
MGSSHHHHHH SSGLVPRGSH MANMHQLLTE LVNRGGSDLH LTTNSPPQIR IDGKLLPLDM PPLNAVDTKQ LCYSILTEQQ KHKFEENNE LDLSFGIKGL SRFRGNVFVQ RGAVAGVFRV IPYKILSFEE LGLPPVVREL AEKPRGLVLV TGPTGSGKST T LAAIIDKI NTDRHEHIVT VEDPIEYLHP HKSCVVNQRE VGADTKSFKN ALKYILRQDP DVVLVGELRD LETIEAALTL AE TGHLCFA TLHTNSAVQT INRIVDVFPS YQQPQVRAQL SFVLEGVLSQ TLLPKASGTG RVLAIEVMVP NPAIRNLIRE DKI HQIYSQ MQVGQEKFGM MTMNQCLYGL LQKRHITMDV GMGRSPDPDE LKQMLTSGVR PQAPRPPMR

UniProtKB: Twitching motility pilus retraction ATPase

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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

Concentration0.5 mg/mL
BufferpH: 8
GridModel: Homemade / Material: GOLD
VitrificationCryogen name: ETHANE-PROPANE

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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: FEI FALCON III (4k x 4k) / Average electron dose: 42.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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

6ojx

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 100786
DetailsFalcon 3EC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6ojx


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial fitting in chimera, flexible fitting in Phenix-refine
RefinementProtocol: FLEXIBLE FIT / Overall B value: 38.4
Output model

PDB-6oll:
CryoEM structure of PilT4 from Geobacter metallireducens without adding nucleotide: C2oocooc conformation

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