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- EMDB-5795: Helical reconstruction of hREGIIIalpha filaments on vesicles -

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Basic information

Entry
Database: EMDB / ID: EMD-5795
TitleHelical reconstruction of hREGIIIalpha filaments on vesicles
Map dataHelical reconstruction of hREGIIIalpha filaments on vesicles
Sample
  • Sample: hREGIIIalpha filament
  • Protein or peptide: RegIIIalpha
KeywordsC-type lectin / membrane permeabilization / pore formation / hexameric pore / innate immunity / bactericidal toxin
Function / homology
Function and homology information


disruption of cell wall in another organism / positive regulation of detection of glucose / response to symbiotic bacterium / positive regulation of keratinocyte proliferation / negative regulation of keratinocyte differentiation / negative regulation of inflammatory response to wounding / oligosaccharide binding / peptidoglycan binding / heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules / Antimicrobial peptides ...disruption of cell wall in another organism / positive regulation of detection of glucose / response to symbiotic bacterium / positive regulation of keratinocyte proliferation / negative regulation of keratinocyte differentiation / negative regulation of inflammatory response to wounding / oligosaccharide binding / peptidoglycan binding / heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules / Antimicrobial peptides / positive regulation of wound healing / acute-phase response / hormone activity / response to peptide hormone / negative regulation of inflammatory response / response to wounding / antimicrobial humoral immune response mediated by antimicrobial peptide / signaling receptor activity / carbohydrate binding / positive regulation of cell population proliferation / extracellular space / extracellular region / identical protein binding / cytoplasm
Similarity search - Function
C-type lectin, conserved site / C-type lectin domain signature. / Lectin C-type domain / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / C-type lectin-like/link domain superfamily / C-type lectin fold
Similarity search - Domain/homology
Regenerating islet-derived protein 3-alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 9.2 Å
AuthorsMukherjee S / Zheng H / Derebe M / Callenberg K / Partch CL / Rollins D / Propheter DC / Rizo J / Grabe M / Jiang Q-X / Hooper LV
CitationJournal: Nature / Year: 2014
Title: Antibacterial membrane attack by a pore-forming intestinal C-type lectin.
Authors: Sohini Mukherjee / Hui Zheng / Mehabaw G Derebe / Keith M Callenberg / Carrie L Partch / Darcy Rollins / Daniel C Propheter / Josep Rizo / Michael Grabe / Qiu-Xing Jiang / Lora V Hooper /
Abstract: Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are ...Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate, but the mechanism by which they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. We show that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. We derive a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and show that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.
History
DepositionNov 15, 2013-
Header (metadata) releaseNov 27, 2013-
Map releaseNov 27, 2013-
UpdateDec 25, 2013-
Current statusDec 25, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5795_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5795.map.gz / Format: CCP4 / Size: 6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical reconstruction of hREGIIIalpha filaments on vesicles
Voxel sizeX=Y=Z: 2.26 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-2.15929651 - 2.73892903
Average (Standard dev.)-0.00271109 (±0.24077381)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100160
Spacing100100160
CellA: 226.0 Å / B: 226.0 Å / C: 361.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.262.262.26
M x/y/z100100160
origin x/y/z0.0000.0000.000
length x/y/z226.000226.000361.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100160
D min/max/mean-2.1592.739-0.003

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Supplemental data

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Sample components

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Entire : hREGIIIalpha filament

EntireName: hREGIIIalpha filament
Components
  • Sample: hREGIIIalpha filament
  • Protein or peptide: RegIIIalpha

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Supramolecule #1000: hREGIIIalpha filament

SupramoleculeName: hREGIIIalpha filament / type: sample / ID: 1000 / Oligomeric state: Three-stranded helix / Number unique components: 1
Molecular weightExperimental: 732 MDa / Theoretical: 880 MDa / Method: Calculated from sequence

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Macromolecule #1: RegIIIalpha

MacromoleculeName: RegIIIalpha / type: protein_or_peptide / ID: 1 / Name.synonym: HIP/PAP
Details: The purified protein was incubated with lipid vesicles to form the filaments.
Number of copies: 1 / Oligomeric state: monomer in solution / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Intestine / Cell: Enterocyte
Molecular weightExperimental: 15 KDa / Theoretical: 15 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21DE3-RIPL / Recombinant plasmid: pET3a
SequenceUniProtKB: Regenerating islet-derived protein 3-alpha

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 5.5 / Details: 10 mM MES, 25 mM NaCl
GridDetails: Quantifoil R2/2 200 mesh holey copper grids were covered with a layer of ultra-thin carbon film (1-3 nm) from the carbon side and were glow-discharged in a Denton Vacuum DV-502A instrument ...Details: Quantifoil R2/2 200 mesh holey copper grids were covered with a layer of ultra-thin carbon film (1-3 nm) from the carbon side and were glow-discharged in a Denton Vacuum DV-502A instrument with a 35 mA current for 60 s in amylamine atmosphere.
VitrificationCryogen name: ETHANE / Chamber humidity: 92 % / Chamber temperature: 95.15 K / Instrument: FEI VITROBOT MARK III
Method: Blot for 2 seconds before plunging into liquid ethane

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Electron microscopy

MicroscopeJEOL 2200FS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 61950 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: FEI / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 35.0 eV
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMin: 93 K / Max: 103 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: corrected at 60,000x magnification
DateDec 27, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 75 / Average electron dose: 20 e/Å2
Details: The Zeiss SCAI scanner can handle six 4 x 5 inches Kodak SO-163 films at one time, so only films with no obvious drift or astigmatism were scanned.
Od range: 1.5 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each filament
Final reconstructionApplied symmetry - Helical parameters - Δz: 18.52 Å
Applied symmetry - Helical parameters - Δ&Phi: 54.21 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.2 Å / Resolution method: OTHER / Software - Name: Spider
Details: Final data were calculated from three separate datasets from three sessions of data collection.
DetailsIHRSR method was used. We could not determine handedness from tilt pairs as our microscope did not allow stable high-quality data collection.

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Atomic model buiding 1

Initial modelPDB ID:

4mth


Chain - Chain ID: A
SoftwareName: Chimera, Situs
DetailsThe docking of one X-ray model into a segmented map corresponding to one subunit was first done manually in Chimera, and then optimized using Situs.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 20.76 / Target criteria: cross-correlation

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