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- PDB-8dit: Cryo-EM structure of a HOPS core complex containing Vps33, Vps16,... -

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Basic information

Entry
Database: PDB / ID: 8dit
TitleCryo-EM structure of a HOPS core complex containing Vps33, Vps16, and Vps18
Components
  • Vacuolar protein sorting-associated protein 16Vacuole
  • Vacuolar protein sorting-associated protein 18Vacuole
  • Vacuolar protein sorting-associated protein 33Vacuole
KeywordsPROTEIN TRANSPORT / HOPS / membrane tethering / SNAREs / membrane fusion / endolysosomal trafficking
Biological speciesChaetomium thermophilum (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsPort, S.A. / Farrell, P.D. / Jeffrey, P.D. / DiMaio, F. / Hughson, F.M.
Funding support Germany, United States, 3items
OrganizationGrant numberCountry
German Research Foundation (DFG)PO2195/1-1 Germany
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071574 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM123089 United States
CitationJournal: To Be Published
Title: Cryo-EM structure of the HOPS core complex and its implication for SNARE assembly
Authors: Port, S.A. / Farrell, P.D. / Jeffrey, P.D. / DiMaio, F. / Hughson, F.M.
History
DepositionJun 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 31, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Vacuolar protein sorting-associated protein 33
B: Vacuolar protein sorting-associated protein 16
C: Vacuolar protein sorting-associated protein 18


Theoretical massNumber of molelcules
Total (without water)279,8523
Polymers279,8523
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, 1:1:1 stoichiometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Vacuolar protein sorting-associated protein 33 / Vacuole


Mass: 77119.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Plasmid: pQLinkH-Vps33:Vps16 / Details (production host): co-expression plasmid / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 codon+
#2: Protein Vacuolar protein sorting-associated protein 16 / Vacuole


Mass: 94204.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Plasmid: pQLinkH-Vps33:Vps16 / Details (production host): co-expression plasmid / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 codon+
#3: Protein Vacuolar protein sorting-associated protein 18 / Vacuole


Mass: 108527.570 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Plasmid: pQLinkN-Vps18 / Production host: Escherichia coli (E. coli) / Variant (production host): C43

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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

Component
IDNameTypeEntity IDParent-IDSource
1HOPS core complexCOMPLEXall0RECOMBINANT
2His-Vps33:Vps16COMPLEX#1-#21RECOMBINANT
3Vps18COMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.280 MDaNO
210.171 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Chaetomium thermophilum (fungus)209285
32Chaetomium thermophilum (fungus)209285
43Chaetomium thermophilum (fungus)209285
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562BL21 codon+pQLinkH-Vps33:Vps16
43Escherichia coli (E. coli)562C43 (DE3)pQLinkN-Vps18
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESHEPES1
2150 mMsodium chlorideNaClSodium chloride1
31 mMDTTDTT1
SpecimenConc.: 0.08 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: PELCO Easiglow, 10 mA/ 10 s discharge/ 30 s hold / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
Details: Blotting parameters: 30 seconds waiting time, blotting force 1, blot total 1

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 2000 nm / Cs: 0.03 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5.6 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3590
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 32 / Used frames/image: 1-32

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4cryoSPARC2.15.0CTF correction
10cryoSPARC2.15.0initial Euler assignment
11cryoSPARCv2.15.0final Euler assignment
12cryoSPARC2.15.0classification
13cryoSPARC2.15.03D reconstruction
14RELION3.1.03D reconstructionpixel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4092244 / Details: blob picker 60-280A (circular)
3D reconstructionResolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154917 / Symmetry type: POINT
RefinementHighest resolution: 5.1 Å

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