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- PDB-7kuh: MicroED structure of mVDAC -

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Basic information

Entry
Database: PDB / ID: 7kuh
TitleMicroED structure of mVDAC
ComponentsVoltage-dependent anion-selective channel protein 1
KeywordsTRANSPORT PROTEIN / Channel / mammalian / voltage dependent
Function / homology
Function and homology information


Pyruvate metabolism / negative regulation of calcium import into the mitochondrion / positive regulation of type 2 mitophagy / voltage-gated monoatomic anion channel activity / PINK1-PRKN Mediated Mitophagy / mitochondrial calcium ion transmembrane transport / neuron-neuron synaptic transmission / acetyl-CoA biosynthetic process from pyruvate / calcium import into the mitochondrion / ceramide binding ...Pyruvate metabolism / negative regulation of calcium import into the mitochondrion / positive regulation of type 2 mitophagy / voltage-gated monoatomic anion channel activity / PINK1-PRKN Mediated Mitophagy / mitochondrial calcium ion transmembrane transport / neuron-neuron synaptic transmission / acetyl-CoA biosynthetic process from pyruvate / calcium import into the mitochondrion / ceramide binding / regulation of mitophagy / mitochondrial permeability transition pore complex / monoatomic anion channel activity / phosphatidylcholine binding / Ub-specific processing proteases / oxysterol binding / lipid transport / porin activity / cholesterol binding / mitochondrial nucleoid / behavioral fear response / negative regulation of reactive oxygen species metabolic process / presynaptic active zone membrane / epithelial cell differentiation / learning / postsynaptic density membrane / synaptic vesicle / myelin sheath / chemical synaptic transmission / mitochondrial outer membrane / transmembrane transporter binding / mitochondrial inner membrane / membrane raft / protein-containing complex binding / negative regulation of apoptotic process / apoptotic process / protein kinase binding / protein-containing complex / mitochondrion / ATP binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
Eukaryotic mitochondrial porin signature. / Porin, eukaryotic type / Eukaryotic porin/Tom40 / Eukaryotic porin / Porin domain superfamily
Similarity search - Domain/homology
Non-selective voltage-gated ion channel VDAC1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 3.12 Å
AuthorsMartynowycz, M.W. / Khan, F. / Hattne, J. / Abramson, J. / Gonen, T.
Funding support United States, 3items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM135175 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel.
Authors: Michael W Martynowycz / Farha Khan / Johan Hattne / Jeff Abramson / Tamir Gonen /
Abstract: A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle ...A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.
History
DepositionNov 25, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Assembly

Deposited unit
X: Voltage-dependent anion-selective channel protein 1


Theoretical massNumber of molelcules
Total (without water)32,1961
Polymers32,1961
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area17250 Å2
Unit cell
Length a, b, c (Å)98.900, 58.210, 69.540
Angle α, β, γ (deg.)90.000, 99.444, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

#1: Protein Voltage-dependent anion-selective channel protein 1 / mVDAC1 / Outer mitochondrial membrane protein porin 1 / Plasmalemmal porin / Voltage-dependent ...mVDAC1 / Outer mitochondrial membrane protein porin 1 / Plasmalemmal porin / Voltage-dependent anion-selective channel protein 5 / mVDAC5


Mass: 32195.879 Da / Num. of mol.: 1 / Mutation: K12E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Vdac1, Vdac5 / Production host: Escherichia coli (E. coli) / References: UniProt: Q60932

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Murine voltage-dependent anion channel mutant / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.03287 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.5
SpecimenConc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse crystal embedded in lipid bicelles
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Data collection

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 5 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 180 / Num. of grids imaged: 2 / Num. of real images: 150
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 3000 mm / Tilt angle list: -60,68
EM diffraction shellResolution: 3.232→3.12 Å / Fourier space coverage: 57.5 % / Multiplicity: 6.2 / Num. of structure factors: 565 / Phase residual: 27 °
EM diffraction statsFourier space coverage: 76.29 % / High resolution: 3.12 Å / Num. of intensities measured: 32723 / Num. of structure factors: 5410 / Phase error: 25 ° / Phase residual: 25 ° / Phase error rejection criteria: none used / Rmerge: 0.47 / Rsym: 0.22
ReflectionBiso Wilson estimate: 93.7 Å2

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Processing

Software
NameVersionClassificationNB
phenix.refine1.18.2_3874+SVNrefinement
PHENIX1.18.2_3874+SVNrefinement
EM software
IDNameVersionCategoryDetails
1EPUimage acquisitionEPU-D
6PHENIX1.18.2model fittingPhaser
8PHENIX1.18.2model refinementphenix.refine
9PHENIXmolecular replacementPhaser
12AIMLESScrystallography merging
13PHENIX3D reconstruction
Image processingDetails: CetaD 2x binned
EM 3D crystal entity∠α: 90 ° / ∠β: 99.44 ° / ∠γ: 90 ° / A: 98.9 Å / B: 58.21 Å / C: 69.54 Å / Space group name: C121 / Space group num: 5
CTF correctionType: NONE
3D reconstructionResolution: 3.12 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Algorithm: FOURIER SPACE / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 90 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Maximum liklihood
Details: Standard refinement with electron scattering factors
Atomic model buildingPDB-ID: 3EMN

3emn


Accession code: 3EMN / Source name: PDB / Type: experimental model
RefinementResolution: 3.12→29.46 Å / SU ML: 0.5567 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 25.42
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: Simple refinement without optimization.
RfactorNum. reflection% reflection
Rfree0.287 266 4.92 %
Rwork0.2565 5144 -
obs0.258 5410 76.4 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 92.73 Å2
Refinement stepCycle: LAST / Resolution: 3.12→29.46 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2167 0 0 0 2167
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0022225
ELECTRON CRYSTALLOGRAPHYf_angle_d0.58543012
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.042333
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.004389
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d11.0242303
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.12-3.930.34711330.28872444ELECTRON CRYSTALLOGRAPHY73.65
3.93-29.460.26581330.24562700ELECTRON CRYSTALLOGRAPHY79.09

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