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- EMDB-26886: Erythrocyte ankyrin-1 complex class 2 local refinement of AQP1 (C... -

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Basic information

Entry
Database: EMDB / ID: EMD-26886
TitleErythrocyte ankyrin-1 complex class 2 local refinement of AQP1 (C4 symmetry applied)
Map dataMain map used for model building/refinement. Density modified and cropped to minimal box using phenix.resolve_cryo_em, resampled on finer grid using relion_image_handler.
Sample
  • Complex: Local refinement of aquaporin 1 in class 2 of ankyrin complex; local C4 symmetry applied.
    • Protein or peptide: Aquaporin-1
  • Ligand: CHOLESTEROL
  • Ligand: water
Function / homology
Function and homology information


metanephric descending thin limb development / metanephric proximal straight tubule development / metanephric proximal convoluted tubule segment 2 development / metanephric glomerulus vasculature development / nitric oxide transmembrane transporter activity / cerebrospinal fluid secretion / lipid digestion / cellular response to salt stress / renal water transport / corticotropin secretion ...metanephric descending thin limb development / metanephric proximal straight tubule development / metanephric proximal convoluted tubule segment 2 development / metanephric glomerulus vasculature development / nitric oxide transmembrane transporter activity / cerebrospinal fluid secretion / lipid digestion / cellular response to salt stress / renal water transport / corticotropin secretion / glycerol transmembrane transporter activity / secretory granule organization / carbon dioxide transmembrane transport / carbon dioxide transmembrane transporter activity / renal water absorption / water transmembrane transporter activity / Passive transport by Aquaporins / positive regulation of saliva secretion / glycerol transmembrane transport / establishment or maintenance of actin cytoskeleton polarity / pancreatic juice secretion / lateral ventricle development / cellular response to mercury ion / potassium ion transmembrane transporter activity / intracellular water homeostasis / intracellularly cGMP-activated cation channel activity / water channel activity / water transport / transepithelial water transport / ammonium transmembrane transport / ammonium channel activity / ankyrin-1 complex / glomerular filtration / camera-type eye morphogenesis / fibroblast migration / multicellular organismal-level water homeostasis / cellular homeostasis / cellular hyperosmotic response / hyperosmotic response / cell volume homeostasis / positive regulation of fibroblast migration / odontogenesis / nitric oxide transport / cGMP-mediated signaling / brush border / transmembrane transporter activity / potassium channel activity / cellular response to nitric oxide / renal water homeostasis / cellular response to retinoic acid / cellular response to cAMP / cellular response to copper ion / sensory perception of pain / ephrin receptor binding / cellular response to dexamethasone stimulus / : / basal plasma membrane / establishment of localization in cell / brush border membrane / carbon dioxide transport / wound healing / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / potassium ion transport / sarcolemma / cellular response to hydrogen peroxide / cellular response to mechanical stimulus / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of angiogenesis / cellular response to UV / positive regulation of fibroblast proliferation / apical part of cell / cellular response to hypoxia / basolateral plasma membrane / nuclear membrane / defense response to Gram-negative bacterium / apical plasma membrane / axon / negative regulation of apoptotic process / extracellular exosome / identical protein binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
Aquaporin 1 / Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Aquaporin-like
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsVallese F / Kim K / Yen LY / Johnston JD / Noble AJ / Cali T / Clarke OB
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Architecture of the human erythrocyte ankyrin-1 complex.
Authors: Francesca Vallese / Kookjoo Kim / Laura Y Yen / Jake D Johnston / Alex J Noble / Tito Calì / Oliver Biggs Clarke /
Abstract: The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association ...The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2, glycophorin A and glycophorin B. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and, unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.
History
DepositionMay 9, 2022-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateJul 27, 2022-
Current statusJul 27, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26886.png

Downloads & links

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Map

FileDownload / File: emd_26886.map.gz / Format: CCP4 / Size: 51.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map used for model building/refinement. Density modified and cropped to minimal box using phenix.resolve_cryo_em, resampled on finer grid using relion_image_handler.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.42 Å/pix.
x 238 pix.
= 98.77 Å		0.42 Å/pix.
x 238 pix.
= 98.77 Å		0.42 Å/pix.
x 238 pix.
= 98.77 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.415 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-2.8086247 - 4.491444
Average (Standard dev.)0.005608914 (±0.35663363)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions238238238
Spacing238238238
CellA=B=C: 98.77 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Mask used for FSC calculation.

Fileemd_26886_additional_1.map
AnnotationMask used for FSC calculation.
Projections & Slices
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Additional map: Half map 1 (not cropped/resampled)

Fileemd_26886_additional_2.map
AnnotationHalf map 1 (not cropped/resampled)
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Additional map: Half map 2 (not cropped/resampled)

Fileemd_26886_additional_3.map
AnnotationHalf map 2 (not cropped/resampled)
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Half map: Half map 2, cropped and resampleed to match main map.

Fileemd_26886_half_map_1.map
AnnotationHalf map 2, cropped and resampleed to match main map.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

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Half map: Half map 1, cropped and resampleed to match main map.

Fileemd_26886_half_map_2.map
AnnotationHalf map 1, cropped and resampleed to match main map.
Projections & Slices
AxesZYX

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Sample components

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Entire : Local refinement of aquaporin 1 in class 2 of ankyrin complex; lo...

EntireName: Local refinement of aquaporin 1 in class 2 of ankyrin complex; local C4 symmetry applied.
Components
  • Complex: Local refinement of aquaporin 1 in class 2 of ankyrin complex; local C4 symmetry applied.
    • Protein or peptide: Aquaporin-1
  • Ligand: CHOLESTEROL
  • Ligand: water

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Supramolecule #1: Local refinement of aquaporin 1 in class 2 of ankyrin complex; lo...

SupramoleculeName: Local refinement of aquaporin 1 in class 2 of ankyrin complex; local C4 symmetry applied.
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human) / Organ: Blood / Tissue: Erythrocytes / Location in cell: Plasma membrane

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Macromolecule #1: Aquaporin-1

MacromoleculeName: Aquaporin-1 / type: protein_or_peptide / ID: 1 / Details: Palmitoylated at Cys-87 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: human (human) / Organ: Blood / Tissue: Erythrocytes
Molecular weightTheoretical: 28.78832 KDa
SequenceString: MASEFKKKLF WRAVVAEFLA TTLFVFISIG SALGFKYPVG NNQTAVQDNV KVSLAFGLSI ATLAQSVGHI SGAHLNPAVT LGLLLS(P1L)QI SIFRALMYII AQCVGAIVAT AILSGITSSL TGNSLGRNDL ADGVNSGQGL GIEIIGTLQL VLCVLAT TD RRRRDLGGSA ...String:
MASEFKKKLF WRAVVAEFLA TTLFVFISIG SALGFKYPVG NNQTAVQDNV KVSLAFGLSI ATLAQSVGHI SGAHLNPAVT LGLLLS(P1L)QI SIFRALMYII AQCVGAIVAT AILSGITSSL TGNSLGRNDL ADGVNSGQGL GIEIIGTLQL VLCVLAT TD RRRRDLGGSA PLAIGLSVAL GHLLAIDYTG CGINPARSFG SAVITHNFSN HWIFWVGPFI GGALAVLIYD FILAPRSS D LTDRVKVWTS GQVEEYDLDA DDINSRVEMK PK

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Macromolecule #2: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 2 / Number of copies: 4 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 113 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration8 mg/mL
BufferpH: 7.4
Details: Final gel filtration buffer contained 0.05 % (w/v) digitonin, 130mM KCl, 20mM HEPES pH 7.4, 1mM ATP, 1mM MgCl2, 1mM PMSF. Peak fractions were concentrated to 8mg/mL, and 0.01% (w/v) of ...Details: Final gel filtration buffer contained 0.05 % (w/v) digitonin, 130mM KCl, 20mM HEPES pH 7.4, 1mM ATP, 1mM MgCl2, 1mM PMSF. Peak fractions were concentrated to 8mg/mL, and 0.01% (w/v) of glycyrrhizic acid was added immediately prior to vitrification.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 4-6 seconds, wait time 30 seconds..
DetailsAnkyrin complex mixture, purified from digitonin-solubilized erythrocyte ghost membranes.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 14464 / Average exposure time: 2.5 sec. / Average electron dose: 58.0 e/Å2 / Details: Two grids were imaged in a single session.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 3)
Details: Patch CTF (cryoSPARC v3) followed by per particle defocus refinement and refinement of higher order aberrations (cryoSPARC v3)
Startup modelType of model: INSILICO MODEL
In silico model: Generated by stochastic gradient descent using ab intio reconstruction job type in cryoSPARC v3.
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 108425
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final 3D classificationDetails: This corresponds to a class of band 3-GPA particles isolated from a mixture mostly consisting of ankyrin complexes.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4csk


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7uze:
Erythrocyte ankyrin-1 complex class 2 local refinement of AQP1 (C4 symmetry applied)

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