National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
R01GM071940
United States
Citation
Journal: Nat Struct Mol Biol / Year: 2022 Title: Structure, dynamics and assembly of the ankyrin complex on human red blood cell membrane. Authors: Xian Xia / Shiheng Liu / Z Hong Zhou / Abstract: The cytoskeleton of a red blood cell (RBC) is anchored to the cell membrane by the ankyrin complex. This complex is assembled during RBC genesis and comprises primarily band 3, protein 4.2 and ...The cytoskeleton of a red blood cell (RBC) is anchored to the cell membrane by the ankyrin complex. This complex is assembled during RBC genesis and comprises primarily band 3, protein 4.2 and ankyrin, whose mutations contribute to numerous human inherited diseases. High-resolution structures of the ankyrin complex have been long sought-after to understand its assembly and disease-causing mutations. Here, we analyzed native complexes on the human RBC membrane by stepwise fractionation. Cryo-electron microscopy structures of nine band-3-associated complexes reveal that protein 4.2 stabilizes the cytoplasmic domain of band 3 dimer. In turn, the superhelix-shaped ankyrin binds to this protein 4.2 via ankyrin repeats (ARs) 6-13 and to another band 3 dimer via ARs 17-20, bridging two band 3 dimers in the ankyrin complex. Integration of these structures with both prior data and our biochemical data supports a model of ankyrin complex assembly during erythropoiesis and identifies interactions essential for the mechanical stability of RBC.
Download / File: emd_26145.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
band 3-protein 4.2 complex in loosely-bound conformation
Voxel size
X=Y=Z: 1.1 Å
Density
Contour Level
By AUTHOR: 0.01
Minimum - Maximum
-0.027682312 - 0.07548089
Average (Standard dev.)
0.00023032457 (±0.0023555094)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
320
320
320
Spacing
320
320
320
Cell
A=B=C: 352.0 Å α=β=γ: 90.0 °
-
Supplemental data
-
Sample components
-
Entire : High-salt fraction 1 from human red blood cell membrane
Entire
Name: High-salt fraction 1 from human red blood cell membrane
Components
Complex: High-salt fraction 1 from human red blood cell membrane
-
Supramolecule #1: High-salt fraction 1 from human red blood cell membrane
Supramolecule
Name: High-salt fraction 1 from human red blood cell membrane type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)
Organism: Homo sapiens (human)
Molecular weight
Theoretical: 280 KDa
-
Experimental details
-
Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
2.5 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Formula
Name
10.0 mM
NH2C(CH2OH)3HCl
Tris-HCl
300.0 mM
NaCl
Sodium Chloride
1.0 mM
HSCH2CH(OH)CH(OH)CH2SH
DTT
0.015 %
C24H46O11
DDM
Grid
Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN KRIOS
Temperature
Min: 100.0 K / Max: 100.0 K
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 20842 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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