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- PDB-3m9i: Electron crystallographic structure of lens Aquaporin-0 (AQP0) (l... -

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Basic information

Entry
Database: PDB / ID: 3m9i
TitleElectron crystallographic structure of lens Aquaporin-0 (AQP0) (lens MIP) in E. coli polar lipids
ComponentsLens fiber major intrinsic protein
KeywordsMEMBRANE PROTEIN / water channel / lens / lipid-protein interactions
Function / homology
Function and homology information


gap junction-mediated intercellular transport / water channel activity / gap junction / water transport / structural constituent of eye lens / 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 / gap junction / water transport / structural constituent of eye lens / response to stimulus / lens development in camera-type eye / positive regulation of cell adhesion / visual perception / protein homotetramerization / calmodulin binding / integral component of plasma membrane / endoplasmic reticulum / plasma membrane
MIP family signature. / Major intrinsic protein / Aquaporin transporter / Aquaporin-like / Major intrinsic protein, conserved site / Major intrinsic protein
Lens fiber major intrinsic protein
Biological speciesOvis aries (sheep)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.5 Å
AuthorsHite, R.K. / Li, Z. / Walz, T.
CitationJournal: EMBO J. / Year: 2010
Title: Principles of membrane protein interactions with annular lipids deduced from aquaporin-0 2D crystals.
Authors: Richard K Hite / Zongli Li / Thomas Walz /
Abstract: We have previously described the interactions of aquaporin-0 (AQP0) with dimyristoyl phosphatidylcholine (DMPC) lipids. We have now determined the 2.5 A structure of AQP0 in two-dimensional (2D) ...We have previously described the interactions of aquaporin-0 (AQP0) with dimyristoyl phosphatidylcholine (DMPC) lipids. We have now determined the 2.5 A structure of AQP0 in two-dimensional (2D) crystals formed with Escherichia coli polar lipids (EPLs), which differ from DMPC both in headgroups and acyl chains. Comparison of the two structures shows that AQP0 does not adapt to the different length of the acyl chains in EPLs and that the distance between the phosphodiester groups in the two leaflets of the DMPC and EPL bilayers is almost identical. The EPL headgroups interact differently with AQP0 than do those of DMPC, but the acyl chains in the EPL and DMPC bilayers occupy similar positions. The interactions of annular lipids with membrane proteins seem to be driven by the propensity of the acyl chains to fill gaps in the protein surface. Interactions of the lipid headgroups may be responsible for the specific interactions found in tightly bound lipids but seem to have a negligible effect on interactions of generic annular lipids with membrane proteins.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMar 22, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 12, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 8, 2017Group: Data collection / Data processing / Refinement description
Category: em_3d_reconstruction / em_image_scans / software

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Assembly

Deposited unit
A: Lens fiber major intrinsic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,7518
Polymers23,5141
Non-polymers5,2367
Water1448
1
A: Lens fiber major intrinsic protein
hetero molecules
x 8


Theoretical massNumber of molelcules
Total (without water)230,00764
Polymers188,1168
Non-polymers41,89256
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation3_555-y,x,z1
crystal symmetry operation4_555y,-x,z1
crystal symmetry operation5_555-x,y,-z1
crystal symmetry operation6_555x,-y,-z1
crystal symmetry operation7_555y,x,-z1
crystal symmetry operation8_555-y,-x,-z1
Buried area55130 Å2
ΔGint-546 kcal/mol
Surface area71290 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)65.500, 65.500, 200.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number89
Space group name H-MP422
DetailsThe octamer can be generated by applying P422 symmetry.

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Components

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


Mass: 23514.447 Da / Num. of mol.: 1 / Fragment: UNP residues 7 to 226 / Source method: isolated from a natural source / Source: (natural) Ovis aries (sheep) / References: UniProt: Q6J8I9
#2: Chemical
ChemComp-3PE / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE / 3-SN-PHOSPHATIDYLETHANOLAMINE


Mass: 748.065 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C41H82NO8P / Comment: phospholipid *YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O / Water

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY / Number of used crystals: 281
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: lens Aquaporin 0 / Type: COMPLEX
Buffer solutionpH: 8
SpecimenDetails: Purified membranes were solubilized in 4% (w/v) octyl glucoside in 10 mM Tris (pH 8.0) for 30 min at 22C
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Crystal growTemperature: 300 K / Method: microdialysis / pH: 6
Details: 100 mM NaCl, 50mM MgCl2, 10mM MES pH 6.0, MICRODIALYSIS, temperature 300K

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Data collection

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingFilm or detector model: GENERIC GATAN (4k x 4k)
DiffractionMean temperature: 279 K
Diffraction sourceSource: ELECTRON MICROSCOPE / Type: OTHER
DetectorType: Gatan / Detector: CCD / Date: Feb 24, 2009
ReflectionResolution: 2.5→15.89 Å / Num. all: 14417 / Num. obs: 14417 / % possible obs: 92.3 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 8.1 % / Biso Wilson estimate: 14.9 Å2 / Rmerge(I) obs: 0.226 / Rsym value: 0.189
Reflection shellResolution: 2.5→2.66 Å / Redundancy: 4 % / Num. unique all: 1935 / % possible all: 83.7

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Processing

Software
NameVersionClassification
GatanDigital Micrographdata collection
PHASERphasing
CNS1.2refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2B6O
Resolution: 2.5→15.89 Å / Rfactor Rfree error: 0.007 / Data cutoff high absF: 2341911.44 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber / Details: BULK SOLVENT MODEL USED
RfactorNum. reflection% reflectionSelection details
Rfree0.284 1453 10.2 %RANDOM
Rwork0.25 ---
All0.25 14254 --
Obs0.25 14254 89.8 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 44.0682 Å2 / ksol: 0.1 e/Å3
Displacement parametersBiso mean: 48.6 Å2
Baniso -1Baniso -2Baniso -3
1--9.84 Å20 Å20 Å2
2---9.84 Å20 Å2
3---19.68 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.45 Å0.38 Å
Luzzati d res low-5 Å
Luzzati sigma a0.62 Å0.55 Å
Refinement stepCycle: LAST / Resolution: 2.5→15.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1668 0 273 8 1949
Refine LS restraints
Refinement-IDTypeDev ideal
ELECTRON CRYSTALLOGRAPHYc_bond_d0.008
ELECTRON CRYSTALLOGRAPHYc_angle_deg1.8
ELECTRON CRYSTALLOGRAPHYc_dihedral_angle_d19.7
ELECTRON CRYSTALLOGRAPHYc_improper_angle_d5.98
Refine LS restraints NCSNCS model details: NONE
LS refinement shellResolution: 2.5→2.66 Å / Rfactor Rfree error: 0.029 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.423 210 9.8 %
Rwork0.377 1935 -
Obs--83.7 %
Xplor file

Refinement-ID: ELECTRON CRYSTALLOGRAPHY

Serial noParam fileTopol file
1protein_rep.paramprotein.top
2water_rep.paramwater.top
33pe.param3pe_lipid.top

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