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- PDB-6x8t: CryoEM structure of the apo-SrpI encapasulin complex from Synecho... -

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Basic information

Entry
Database: PDB / ID: 6x8t
TitleCryoEM structure of the apo-SrpI encapasulin complex from Synechococcus elongatus PCC 7942
ComponentsProtein SrpI
KeywordsVIRUS LIKE PARTICLE / encapsulin / nanocompartment / cysteine desulfurase / HK97-fold
Function / homologyType 2A encapsulin shell protein SrpI-like / : / Type 2A encapsulin shell protein SrpI-like / encapsulin nanocompartment / Type 2A encapsulin shell protein SrpI
Function and homology information
Biological speciesSynechococcus elongatus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLaFrance, B.J. / Nichols, R.J. / Phillips, N.R. / Oltrogge, L.M. / Valentin-Alvarado, L.E. / Bischoff, A.J. / Savage, D.F. / Nogales, E.
Funding support United States, 2items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE- 638 SC00016240 United States
National Science Foundation (NSF, United States)1106400 United States
Citation
Journal: Elife / Year: 2021
Title: Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism.
Authors: Robert J Nichols / Benjamin LaFrance / Naiya R Phillips / Devon R Radford / Luke M Oltrogge / Luis E Valentin-Alvarado / Amanda J Bischoff / Eva Nogales / David F Savage /
Abstract: Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies ...Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.
#1: Journal: Biorxiv / Year: 2020
Title: Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism
Authors: Nichols, R.J. / LaFrance, B.J. / Phillips, N.R. / Oltrogge, L.M. / Valentin-Alvarado, L.E. / Bischoff, A.J. / Nogales, E. / Savage, D.F.
History
DepositionJun 1, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 21, 2021Group: Database references / Category: citation / citation_author
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_struct_oper_list
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Protein SrpI


Theoretical massNumber of molelcules
Total (without water)35,3711
Polymers35,3711
Non-polymers00
Water0
1
A: Protein SrpI
x 60


Theoretical massNumber of molelcules
Total (without water)2,122,26760
Polymers2,122,26760
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Protein SrpI
x 5


  • icosahedral pentamer
  • 177 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)176,8565
Polymers176,8565
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Protein SrpI
x 6


  • icosahedral 23 hexamer
  • 212 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)212,2276
Polymers212,2276
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Protein SrpI / SrpI encapsulin shell protein


Mass: 35371.121 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (strain PCC 7942 / FACHB-805) (bacteria)
Strain: PCC 7942 / FACHB-805 / Gene: srpI, Synpcc7942_B2662, pANL38 / Plasmid: pET14 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): LOBSTR / References: UniProt: Q55032

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: SrpI encapsulin apo complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 2.1 MDa / Experimental value: NO
Source (natural)Organism: Synechococcus elongatus (strain PCC 7942 / FACHB-805) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: LOBSTR / Plasmid: pET14
Buffer solutionpH: 10
SpecimenConc.: 2.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K
Details: blot force 5, 3 sec blot, 100 humidity, 4C, 1 sec drain time

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Details: Residual beam tilt corrected in RELION/3.1. CTF refinement performed in RELION/3.1.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.6 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2966
Image scansMovie frames/image: 33

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Processing

SoftwareName: PHENIX / Version: 1.18_3845: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEMimage acquisition
4CTFFIND4.1.10CTF correction
7PHENIX1.18_3845model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
13PHENIX1.18_3845model refinement
Image processingDetails: Images collected in super-resolution mode
CTF correctionDetails: CTF refinement was performed in Relion/3.1 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 57431 / Details: Relion LoG autopicker to select particles
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34425 / Symmetry type: POINT
Atomic model buildingB value: 20 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Details: One subunit was built completely de novo based on sequence/density, and iteratively real space refined with neighboring subunits using PHENIX.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0072271
ELECTRON MICROSCOPYf_angle_d0.6633095
ELECTRON MICROSCOPYf_dihedral_angle_d17.865846
ELECTRON MICROSCOPYf_chiral_restr0.05358
ELECTRON MICROSCOPYf_plane_restr0.006400

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