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- PDB-2b6o: Electron crystallographic structure of lens Aquaporin-0 (AQP0) (l... -

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Basic information

Entry
Database: PDB / ID: 2b6o
TitleElectron crystallographic structure of lens Aquaporin-0 (AQP0) (lens MIP) at 1.9A resolution, in a closed pore state
ComponentsLens fiber major intrinsic protein
KeywordsMEMBRANE PROTEIN / aquaporin-0 junctions / AQP0 / lens MIP / lipid-protein interactions / membrane / lipid bilayer / closed water pore
Function / homology
Function and homology information


gap junction-mediated intercellular transport / water channel activity / water transport / structural constituent of eye lens / gap junction / response to stimulus / lens development in camera-type eye / positive regulation of cell adhesion / visual perception / protein homotetramerization ...gap junction-mediated intercellular transport / water channel activity / water transport / structural constituent of eye lens / gap junction / response to stimulus / lens development in camera-type eye / positive regulation of cell adhesion / visual perception / protein homotetramerization / calmodulin binding / endoplasmic reticulum / plasma membrane
Similarity search - Function
Glycerol uptake facilitator protein / Glycerol uptake facilitator protein. / Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Aquaporin-like / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / Lens fiber major intrinsic protein
Similarity search - Component
Biological speciesOvis aries (sheep)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 1.9 Å
AuthorsGonen, T. / Cheng, Y. / Sliz, P. / Hiroaki, Y. / Fujiyoshi, Y. / Harrison, S.C. / Walz, T.
CitationJournal: Nature / Year: 2005
Title: Lipid-protein interactions in double-layered two-dimensional AQP0 crystals.
Authors: Tamir Gonen / Yifan Cheng / Piotr Sliz / Yoko Hiroaki / Yoshinori Fujiyoshi / Stephen C Harrison / Thomas Walz /
Abstract: Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by ...Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.
History
DepositionOct 3, 2005Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 6, 2005Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection
Category: em_image_scans / em_single_particle_entity / em_software
Item: _em_software.image_processing_id
Revision 1.4Jan 22, 2020Group: Advisory / Derived calculations
Category: pdbx_distant_solvent_atoms / pdbx_struct_assembly ...pdbx_distant_solvent_atoms / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_oper_list
Item: _pdbx_struct_assembly_prop.biol_id
Revision 1.5Jun 17, 2020Group: Advisory / Database references / Category: pdbx_database_remark / struct_ref
Item: _pdbx_database_remark.text / _struct_ref.pdbx_seq_one_letter_code
Revision 1.6Aug 23, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_remark / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_remark.text / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 240EXPERIMENT TYPE : ELECTRON DIFFRACTION DATE OF DATA COLLECTION : 01-DEC-03 TEMPERATURE (KELVIN) : ...EXPERIMENT TYPE : ELECTRON DIFFRACTION DATE OF DATA COLLECTION : 01-DEC-03 TEMPERATURE (KELVIN) : 300.0 PH : 6.00 NUMBER OF CRYSTALS USED : 286 RADIATION SOURCE : JEM3000SFF OPTICS : CRYSTALS TILTED TO MAX : 71.3 DEGREES DETECTOR TYPE : CCD DETECTOR MANUFACTURER : GATAN 2K X 2K DATA SCALING SOFTWARE : GATAN ACCELERATION VOLTAGE (KV) : 300 NUMBER OF UNIQUE REFLECTIONS : 22293 RESOLUTION RANGE HIGH (A) : 1.9 RESOLUTION RANGE LOW (A) : 20.000 OVERALL. COMPLETENESS FOR RANGE (%) : 80.0 DATA REDUNDANCY : 5.700 IN THE HIGHEST RESOLUTION SHELL. HIGHEST RESOLUTION SHELL, RANGE HIGH (A) : 1.90 HIGHEST RESOLUTION SHELL, RANGE LOW (A) : 2.0 COMPLETENESS FOR SHELL (%) : 82.0 DATA REDUNDANCY IN SHELL : 5.70 R MERGE FOR SHELL (I) : 0.166 METHOD USED TO DETERMINE THE STRUCTURE: MOLECULAR : REPLACEMENT SOFTWARE USED: CNS STARTING MODEL: PDB ENTRY 1SOR

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Assembly

Deposited unit
A: Lens fiber major intrinsic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,38610
Polymers28,2851
Non-polymers6,1019
Water1,42379
1
A: Lens fiber major intrinsic protein
hetero molecules
x 8


Theoretical massNumber of molelcules
Total (without water)275,09080
Polymers226,2798
Non-polymers48,81172
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-x+1,-y+1,z1
crystal symmetry operation3_655-y+1,x,z1
crystal symmetry operation4_565y,-x+1,z1
crystal symmetry operation5_655-x+1,y,-z1
crystal symmetry operation6_565x,-y+1,-z1
crystal symmetry operation7_555y,x,-z1
crystal symmetry operation8_665-y+1,-x+1,-z1
Buried area82930 Å2
ΔGint-755 kcal/mol
Surface area72410 Å2
MethodPISA
Unit cell
Length a, b, c (Å)65.500, 65.500, 160.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number89
Space group name H-MP422

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Components

#1: Protein Lens fiber major intrinsic protein / / Aquaporin-0


Mass: 28284.885 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Ovis aries (sheep) / References: UniProt: Q6J8I9
#2: Chemical
ChemComp-MC3 / 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE


Mass: 677.933 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C36H72NO8P / Comment: phospholipid*YM
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 79 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY / Number of used crystals: 286
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: aquaporin-0 / Type: COMPLEX
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: MOLYBDENUM
EM embeddingDetails: 10% trehalose / Material: trehalose
VitrificationCryogen name: NITROGEN
CrystalDensity Matthews: 3.03 Å3/Da / Density % sol: 59.44 %
Crystal growTemperature: 300 K / Method: microdialysis / pH: 6
Details: 20mM MES pH 6.0, 50mM MgCl2, 5mM DTT, MICRODIALYSIS, temperature 300K

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Data collection

MicroscopyModel: JEOL 3000SFF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: HELIUM / Specimen holder model: JEOL
Image recordingFilm or detector model: GENERIC GATAN (4k x 4k)
DiffractionMean temperature: 281 K
Diffraction sourceSource: ELECTRON MICROSCOPE / Type: JEM3000SFF
DetectorType: GATAN / Detector: CCD / Date: Dec 1, 2003 / Details: 4k X 4k
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthRelative weight: 1
ReflectionResolution: 1.9→20 Å / % possible obs: 80 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 5.7 % / Biso Wilson estimate: 18.1 Å2 / Rmerge(I) obs: 0.166
Reflection shellResolution: 1.9→2 Å / Redundancy: 2.5 % / % possible all: 70.5

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Processing

Software
NameClassification
GATANdata collection
GATANdata reduction
CNSrefinement
GATANdata scaling
CNSphasing
EM software
IDNameVersionCategory
1CNS1.1model fitting
2MRC3D reconstruction
3D reconstructionSymmetry type: 2D CRYSTAL
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1SOR - aquaporin-0 (MIP) strructure determined by electron crystallography

1sor


Resolution: 1.9→5 Å / Rfactor Rfree error: 0.008 / Data cutoff high absF: 2606056.45 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.299 1580 9.8 %RANDOM
Rwork0.258 ---
all-16180 --
obs-14600 --
Displacement parametersBiso mean: 58.4 Å2
Baniso -1Baniso -2Baniso -3
1--7.32 Å20 Å20 Å2
2---7.32 Å20 Å2
3---14.64 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.42 Å0.37 Å
Luzzati d res low-5 Å
Luzzati sigma a0.65 Å0.63 Å
Refinement stepCycle: LAST / Resolution: 1.9→5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1783 0 349 79 2211
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
ELECTRON CRYSTALLOGRAPHYo_bond_d0.016
ELECTRON CRYSTALLOGRAPHYo_angle_deg1.9
ELECTRON CRYSTALLOGRAPHYo_dihedral_angle_d19.8
ELECTRON CRYSTALLOGRAPHYo_improper_angle_d1.25
ELECTRON CRYSTALLOGRAPHYo_mcbond_it1.531.5
ELECTRON CRYSTALLOGRAPHYo_mcangle_it2.612
ELECTRON CRYSTALLOGRAPHYo_scbond_it1.632
ELECTRON CRYSTALLOGRAPHYo_scangle_it2.442.5
Xplor file
Refine-IDSerial noParam fileTopol file
ELECTRON CRYSTALLOGRAPHY1protein_rep.paramprotein.top
ELECTRON CRYSTALLOGRAPHY2water_rep.paramwater.top
ELECTRON CRYSTALLOGRAPHY3dmpc.paramdmpc_all_final.top

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