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- EMDB-26917: Protein 4.2 (local refinement from consensus reconstruction of an... -

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Basic information

Entry
Database: EMDB / ID: EMD-26917
TitleProtein 4.2 (local refinement from consensus reconstruction of ankyrin complex classes)
Map dataMain map used for model building/refinement. Density modified and cropped using phenix.resolve_cryo_em, resampled to better visualize high resolution features using relion_image_handler.
Sample
  • Complex: Ankyrin complex mixture purified from digitonin-solubilized erythrocyte ghost membranes
    • Protein or peptide: Protein 4.2
  • Ligand: water
KeywordsMembrane Protein / Anion Exchange / Erythrocyte / Glycoprotein / TRANSPORT PROTEIN
Function / homology
Function and homology information


hemoglobin metabolic process / protein-glutamine gamma-glutamyltransferase activity / ankyrin-1 complex / cortical cytoskeleton / erythrocyte maturation / spleen development / cell morphogenesis / structural constituent of cytoskeleton / multicellular organismal-level iron ion homeostasis / regulation of cell shape ...hemoglobin metabolic process / protein-glutamine gamma-glutamyltransferase activity / ankyrin-1 complex / cortical cytoskeleton / erythrocyte maturation / spleen development / cell morphogenesis / structural constituent of cytoskeleton / multicellular organismal-level iron ion homeostasis / regulation of cell shape / cytoskeleton / ATP binding / plasma membrane
Similarity search - Function
Transglutaminase, N-terminal / Transglutaminase, C-terminal / Transglutaminase, active site / Protein-glutamine gamma-glutamyltransferase, animal / Transglutaminase, C-terminal domain superfamily / : / Transglutaminase family / Transglutaminase family, C-terminal ig like domain / Transglutaminases active site. / Transglutaminase-like superfamily ...Transglutaminase, N-terminal / Transglutaminase, C-terminal / Transglutaminase, active site / Protein-glutamine gamma-glutamyltransferase, animal / Transglutaminase, C-terminal domain superfamily / : / Transglutaminase family / Transglutaminase family, C-terminal ig like domain / Transglutaminases active site. / Transglutaminase-like superfamily / Transglutaminase/protease-like homologues / Transglutaminase-like / Transglutaminase-like superfamily / Papain-like cysteine peptidase superfamily / Immunoglobulin E-set / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 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-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26917.png

Downloads & links

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Map

FileDownload / File: emd_26917.map.gz / Format: CCP4 / Size: 80.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map used for model building/refinement. Density modified and cropped using phenix.resolve_cryo_em, resampled to better visualize high resolution features using relion_image_handler.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.42 Å/pix.
x 276 pix.
= 114.54 Å		0.42 Å/pix.
x 276 pix.
= 114.54 Å		0.42 Å/pix.
x 276 pix.
= 114.54 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-3.603282 - 6.8048925
Average (Standard dev.)0.0014980555 (±0.3272668)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions276276276
Spacing276276276
CellA=B=C: 114.54 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Half map 1 (unmodified)

Fileemd_26917_additional_1.map
AnnotationHalf map 1 (unmodified)
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: Half map 2 (unmodified)

Fileemd_26917_additional_2.map
AnnotationHalf map 2 (unmodified)
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Mask used for FSC calculation.

Fileemd_26917_additional_3.map
AnnotationMask used for FSC calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened (B=56.5) map.

Fileemd_26917_additional_4.map
AnnotationSharpened (B=56.5) map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 (cropped and resampled).

Fileemd_26917_half_map_1.map
AnnotationHalf map 1 (cropped and resampled).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 (cropped and resampled).

Fileemd_26917_half_map_2.map
AnnotationHalf map 2 (cropped and resampled).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ankyrin complex mixture purified from digitonin-solubilized eryth...

EntireName: Ankyrin complex mixture purified from digitonin-solubilized erythrocyte ghost membranes
Components
  • Complex: Ankyrin complex mixture purified from digitonin-solubilized erythrocyte ghost membranes
    • Protein or peptide: Protein 4.2
  • Ligand: water

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Supramolecule #1: Ankyrin complex mixture purified from digitonin-solubilized eryth...

SupramoleculeName: Ankyrin complex mixture purified from digitonin-solubilized erythrocyte ghost membranes
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Protein 4.2 (local refinement from consensus refinement of ankyrin complex)
Source (natural)Organism: Homo sapiens (human) / Organ: Blood / Tissue: Erythrocytes / Location in cell: Plasma membrane

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Macromolecule #1: Protein 4.2

MacromoleculeName: Protein 4.2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Organ: Blood / Tissue: Erythrocytes
Molecular weightTheoretical: 77.096914 KDa
SequenceString: MGQALGIKSC DFQAARNNEE HHTKALSSRR LFVRRGQPFT IILYFRAPVR AFLPALKKVA LTAQTGEQPS KINRTQATFP ISSLGDRKW WSAVVEERDA QSWTISVTTP ADAVIGHYSL LLQVSGRKQL LLGQFTLLFN PWNREDAVFL KNEAQRMEYL L NQNGLIYL ...String:
MGQALGIKSC DFQAARNNEE HHTKALSSRR LFVRRGQPFT IILYFRAPVR AFLPALKKVA LTAQTGEQPS KINRTQATFP ISSLGDRKW WSAVVEERDA QSWTISVTTP ADAVIGHYSL LLQVSGRKQL LLGQFTLLFN PWNREDAVFL KNEAQRMEYL L NQNGLIYL GTADCIQAES WDFGQFEGDV IDLSLRLLSK DKQVEKWSQP VHVARVLGAL LHFLKEQRVL PTPQTQATQE GA LLNKRRG SVPILRQWLT GRGRPVYDGQ AWVLAAVACT VLRCLGIPAR VVTTFASAQG TGGRLLIDEY YNEEGLQNGE GQR GRIWIF QTSTECWMTR PALPQGYDGW QILHPSAPNG GGVLGSCDLV PVRAVKEGTL GLTPAVSDLF AAINASCVVW KCCE DGTLE LTDSNTKYVG NNISTKGVGS DRCEDITQNY KYPEGSLQEK EVLERVEKEK MEREKDNGIR PPSLETASPL YLLLK APSS LPLRGDAQIS VTLVNHSEQE KAVQLAIGVQ AVHYNGVLAA KLWRKKLHLT LSANLEKIIT IGLFFSNFER NPPENT FLR LTAMATHSES NLSCFAQEDI AICRPHLAIK MPEKAEQYQP LTASVSLQNS LDAPMEDCVI SILGRGLIHR ERSYRFR SV WPENTMCAKF QFTPTHVGLQ RLTVEVDCNM FQNLTNYKSV TVVAPELSA

UniProtKB: Protein 4.2

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 193 / 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, 130 mM KCl, 20 mM HEPES, pH 7.4, 1 mM ATP, 1 mM MgCl2, 1 mM PMSF. Peak fractions were concentrated to 8 mg/mL and 0.01% ( w/v ...Details: Final gel filtration buffer contained 0.05% w/v digitonin, 130 mM KCl, 20 mM HEPES, pH 7.4, 1 mM ATP, 1 mM MgCl2, 1 mM PMSF. Peak fractions were concentrated to 8 mg/mL and 0.01% ( w/v 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

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: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 710437
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4yzf


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7uzs:
Protein 4.2 (local refinement from consensus reconstruction of ankyrin complex classes)

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