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- PDB-8ofx: Molecular Mechanism of trypanosomal AQP2 -

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Basic information

Entry
Database: PDB / ID: 8ofx
TitleMolecular Mechanism of trypanosomal AQP2
ComponentsAquaglyceroporin 2
KeywordsMEMBRANE PROTEIN / Aquaporin / Tetramer / Drug Uptake / Glycerol
Function / homology
Function and homology information


glycerol channel activity / urea transmembrane transporter activity / urea transmembrane transport / glycerol transmembrane transport / water channel activity / water transport / membrane
Similarity search - Function
: / Major intrinsic protein / Major intrinsic protein / Aquaporin-like
Similarity search - Domain/homology
: / Aquaglyceroporin-2
Similarity search - Component
Biological speciesTrypanosoma brucei brucei (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWeyand, S.N. / Matusevicius, M. / Yamashita, K.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust101234/Z/13/Z United Kingdom
CitationJournal: To Be Published
Title: Molecular Mechanism of trypanosomal AQP2
Authors: Weyand, S.N.
History
DepositionMar 17, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Aquaglyceroporin 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,0132
Polymers33,6151
Non-polymers3981
Water00
1
A: Aquaglyceroporin 2
hetero molecules

A: Aquaglyceroporin 2
hetero molecules

A: Aquaglyceroporin 2
hetero molecules

A: Aquaglyceroporin 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)136,0528
Polymers134,4594
Non-polymers1,5934
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
point symmetry operation3
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-1), (1), (1)333.44
3generate(-1), (-1), (1)333.44, 333.44
4generate(1), (-1), (1)333.44

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Components

#1: Protein Aquaglyceroporin 2


Mass: 33614.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Melarsoprol / Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Gene: aqp2 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6ZXT3
#2: Chemical ChemComp-VO6 / [(2~{R},4~{S})-2-[4-[[4,6-bis(azanyl)-1,3,5-triazin-2-yl]amino]phenyl]-1,3,2-dithiarsolan-4-yl]methanol


Mass: 398.339 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H15AsN6OS2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Aquaporin 2 tetramer wildtype / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: YES
Source (natural)Organism: Trypanosoma brucei (eukaryote)
Source (recombinant)Organism: Spodoptera (butterflies/moths)
Buffer solutionpH: 7.2 / Details: 20 mM HEPES 100 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium ChlorideNaCl1
220 mMHEPESC8H18N2O4S1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

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: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 56 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0403 / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4GctfCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 100075
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126551 / Algorithm: FOURIER SPACE / Num. of class averages: 6 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
RefinementResolution: 3.2→3.2 Å / Cor.coef. Fo:Fc: 0.9 / SU B: 11.232 / SU ML: 0.194 / ESU R: 0.189
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.32138 --
obs0.32138 55785 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 137.031 Å2
Refinement stepCycle: 1 / Total: 1852
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.0111911
ELECTRON MICROSCOPYr_bond_other_d00.0161811
ELECTRON MICROSCOPYr_angle_refined_deg1.9031.6252610
ELECTRON MICROSCOPYr_angle_other_deg0.611.5474135
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.6375243
ELECTRON MICROSCOPYr_dihedral_angle_2_deg6.953
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.53410255
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0990.2291
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.022201
ELECTRON MICROSCOPYr_gen_planes_other0.0040.02464
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it19.87612.784975
ELECTRON MICROSCOPYr_mcbond_other19.86812.778975
ELECTRON MICROSCOPYr_mcangle_it26.69123.1791217
ELECTRON MICROSCOPYr_mcangle_other26.71323.1991218
ELECTRON MICROSCOPYr_scbond_it24.34814.607936
ELECTRON MICROSCOPYr_scbond_other24.33614.61937
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other33.11926.0261394
ELECTRON MICROSCOPYr_long_range_B_refined39.968312.4931779
ELECTRON MICROSCOPYr_long_range_B_other39.967312.4831780
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.856 4079 -
obs--100 %

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