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- PDB-6mzy: Cryo-EM structure of the HO BMC shell: Icosahedral reconstruction... -

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Basic information

Entry
Database: PDB / ID: 6mzy
TitleCryo-EM structure of the HO BMC shell: Icosahedral reconstruction of the compacted subpopulation
Components
  • (Microcompartments proteinBacterial microcompartment) x 2
  • Ethanolamine utilization protein EutN/carboxysome structural protein Ccml
KeywordsSTRUCTURAL PROTEIN / microcompartment / shell / compartmentalization / BMC fold
Function / homologyMicrocompartment protein, bacteria / Ethanolamine utilization protein EutN/carboxysome structural protein Ccml / Bacterial microcompartments protein, conserved site / EutN/Ccml superfamily / CcmK-like superfamily / BMC domain / Ethanolamine utilisation protein EutN/carboxysome / Bacterial microcompartiments proteins signature. / Ethanolamine utilization protein EutN/carboxysome structural protein Ccml / Microcompartments protein / Microcompartments protein
Function and homology information
Specimen sourceHaliangium ochraceum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.3 Å resolution
AuthorsGreber, B.J. / Sutter, M. / Kerfeld, C.A.
Funding supportUnited States , 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases5 R01 AI114975-05United States
Department of Energy (United States)DE-FG02-91ER20021United States
CitationJournal: Structure / Year: 2019
Title: The Plasticity of Molecular Interactions Governs Bacterial Microcompartment Shell Assembly.
Authors: Basil J Greber / Markus Sutter / Cheryl A Kerfeld
Abstract: Bacterial microcompartments (BMCs) are composed of an enzymatic core encapsulated by a selectively permeable protein shell that enhances catalytic efficiency. Many pathogenic bacteria derive ...Bacterial microcompartments (BMCs) are composed of an enzymatic core encapsulated by a selectively permeable protein shell that enhances catalytic efficiency. Many pathogenic bacteria derive competitive advantages from their BMC-based catabolism, implicating BMCs as drug targets. BMC shells are of interest for bioengineering due to their diverse and selective permeability properties and because they self-assemble. A complete understanding of shell composition and organization is a prerequisite for biotechnological applications. Here, we report the cryoelectron microscopy structure of a BMC shell at 3.0-Å resolution, using an image-processing strategy that allowed us to determine the previously uncharacterized structural details of the interactions formed by the BMC-T and BMC-T shell subunits in the context of the assembled shell. We found unexpected structural plasticity among these interactions, resulting in distinct shell populations assembled from varying numbers of the BMC-T and BMC-T subunits. We discuss the implications of these findings on shell assembly and function.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 6, 2018 / Release: Mar 13, 2019

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
  • Imaged by Jmol
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  • Biological unit as icosahedral pentamer
  • Imaged by Jmol
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  • Biological unit as icosahedral 23 hexamer
  • Imaged by Jmol
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-9310
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  • Superimposition on EM map
  • EMDB-9310
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A1: Ethanolamine utilization protein EutN/carboxysome structural protein Ccml
A2: Microcompartments protein
A3: Microcompartments protein
A4: Microcompartments protein
A5: Microcompartments protein
A6: Microcompartments protein
A7: Microcompartments protein
A8: Microcompartments protein
A9: Microcompartments protein


Theoretical massNumber of molelcules
Total (without water)116,4359
Polyers116,4359
Non-polymers00
Water0
1
A1: Ethanolamine utilization protein EutN/carboxysome structural protein Ccml
A2: Microcompartments protein
A3: Microcompartments protein
A4: Microcompartments protein
A5: Microcompartments protein
A6: Microcompartments protein
A7: Microcompartments protein
A8: Microcompartments protein
A9: Microcompartments protein
x 60


Theoretical massNumber of molelcules
Total (without water)6,986,094540
Polyers6,986,094540
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A1: Ethanolamine utilization protein EutN/carboxysome structural protein Ccml
A2: Microcompartments protein
A3: Microcompartments protein
A4: Microcompartments protein
A5: Microcompartments protein
A6: Microcompartments protein
A7: Microcompartments protein
A8: Microcompartments protein
A9: Microcompartments protein
x 5


  • icosahedral pentamer
  • 582 kDa, 45 polymers
Theoretical massNumber of molelcules
Total (without water)582,17545
Polyers582,17545
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A1: Ethanolamine utilization protein EutN/carboxysome structural protein Ccml
A2: Microcompartments protein
A3: Microcompartments protein
A4: Microcompartments protein
A5: Microcompartments protein
A6: Microcompartments protein
A7: Microcompartments protein
A8: Microcompartments protein
A9: Microcompartments protein
x 6


  • icosahedral 23 hexamer
  • 699 kDa, 54 polymers
Theoretical massNumber of molelcules
Total (without water)698,60954
Polyers698,60954
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1

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Components

#1: Protein/peptide Ethanolamine utilization protein EutN/carboxysome structural protein Ccml


Mass: 9866.319 Da / Num. of mol.: 1
Source: (gene. exp.) Haliangium ochraceum (strain DSM 14365 / JCM 11303 / SMP-2) (bacteria)
Strain: DSM 14365 / JCM 11303 / SMP-2 / Gene: Hoch_5814 / Production host: Escherichia coli (E. coli) / References: UniProt: D0LHE5
#2: Protein/peptide
Microcompartments protein / Bacterial microcompartment


Mass: 10126.718 Da / Num. of mol.: 6
Source: (gene. exp.) Haliangium ochraceum (strain DSM 14365 / JCM 11303 / SMP-2) (bacteria)
Strain: DSM 14365 / JCM 11303 / SMP-2 / Gene: Hoch_5815 / Production host: Escherichia coli (E. coli) / References: UniProt: D0LID5
#3: Protein/peptide Microcompartments protein / Bacterial microcompartment


Mass: 22904.137 Da / Num. of mol.: 2
Source: (gene. exp.) Haliangium ochraceum (strain DSM 14365 / JCM 11303 / SMP-2) (bacteria)
Strain: DSM 14365 / JCM 11303 / SMP-2 / Gene: Hoch_5816 / Production host: Escherichia coli (E. coli) / References: UniProt: D0LID6

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Bacterial microcompartment shell from Haliangium ochraceum
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1,2,3 / Source: RECOMBINANT
Molecular weightValue: 6.5 MDa / Experimental value: NO
Source (natural)Organism: Haliangium ochraceum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer ID
120 mMTris-HCl1
250 mMSodium chlorideNaCl1
30.01 %NP-40 substitute1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins
Details: 5-7 sec incubation of the sample on the grid before blotting and plunging

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Electron microscopy imaging

MicroscopyMicroscope model: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 48543 / Calibrated defocus min: 1000 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm / C2 aperture diameter: 5 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 4.5 sec. / Electron dose: 25 e/Å2
Details: 928 images retained after inspection for image quality. Movie frames were aligned and dose weighed using Motioncor2.
Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 928
Image scansSampling size: 5 microns / Width: 3838 / Height: 3710 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
1RELION1.4particle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION1.4initial Euler assignment
11FREALIGN9final Euler assignment
12RELION1.4classification
13FREALIGN93D reconstruction
CTF correctionDetails: Initial CTF fitting using CTFFIND4, CTF correction applied within RELION.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: 1000 particles were picked manually to generate reference templates for subsequent auto-picking in RELION 1.4.
Number of particles selected: 31800
SymmetryPoint symmetry: I
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 2276 / Algorithm: FOURIER SPACE
Details: Frequency-limited refinement in FREALIGN. To avoid overfitting, resolutions higher than 3.6 Angstroms were excluded from the refinement. Icosahedral symmetry was imposed throughout the refinement procedure.
Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: REAL
Atomic model buildingPDB-ID: 5V74

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