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- EMDB-4631: A dimer component of alpha-1 antitrypsin polymers isolated from Z... -

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Entry
Database: EMDB / ID: EMD-4631
TitleA dimer component of alpha-1 antitrypsin polymers isolated from ZZ explant liver tissue and decorated with Fab 4B12 (component B with ~90 degree rotation around the dimer axis)
Map dataUnmasked dimer component of alpha-1 antitrypsin polymers isolated from explant liver tissue; subunits rotated ~90 degrees around dimer axis. Partially resolved third subunit evident.
Sample
  • Tissue: Polymers of the severe deficiency Z variant of alpha-1 antitrypsin labelled with 4B12 Fab fragments
    • Complex: Complex between two 4B12 Fab fragments and a dimer component of polymers of the severe deficiency Z alpha-1 antitrypsin variant
      • Protein or peptide: Alpha-1 antitrypsin
      • Protein or peptide: 4B12 Fab heavy chain
      • Protein or peptide: 4B12 Fab light chain
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 24.8 Å
AuthorsFaull SV / Elliston ELK / Orlova EV / Gooptu B / Lomas DA / Irving JA
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MR/N024842/1 United Kingdom
Other governmentUCLH/NIHR Biomedical Research Centre United Kingdom
Wellcome TrustWellcome Trust 4-year PhD Interdisciplinary Programme in Structural, Computational and Chemical Biology United Kingdom
CitationJournal: Sci Adv / Year: 2020
Title: The structural basis for Z α-antitrypsin polymerization in the liver.
Authors: Sarah V Faull / Emma L K Elliston / Bibek Gooptu / Alistair M Jagger / Ibrahim Aldobiyan / Adam Redzej / Magd Badaoui / Nina Heyer-Chauhan / S Tamir Rashid / Gary M Reynolds / David H Adams ...Authors: Sarah V Faull / Emma L K Elliston / Bibek Gooptu / Alistair M Jagger / Ibrahim Aldobiyan / Adam Redzej / Magd Badaoui / Nina Heyer-Chauhan / S Tamir Rashid / Gary M Reynolds / David H Adams / Elena Miranda / Elena V Orlova / James A Irving / David A Lomas /
Abstract: The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α-Antitrypsin deficiency is the archetypal ...The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α-antitrypsin as "polymer" chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α-antitrypsin.
History
DepositionFeb 22, 2019-
Header (metadata) releaseMar 25, 2020-
Map releaseMar 25, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4631.png

Downloads & links

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Map

FileDownload / File: emd_4631.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnmasked dimer component of alpha-1 antitrypsin polymers isolated from explant liver tissue; subunits rotated ~90 degrees around dimer axis. Partially resolved third subunit evident.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.54 Å/pix.
x 150 pix.
= 231. Å		1.54 Å/pix.
x 150 pix.
= 231. Å		1.54 Å/pix.
x 150 pix.
= 231. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.54 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.006 / Movie #1: 0.006
Minimum - Maximum-0.013678535 - 0.03558241
Average (Standard dev.)0.0002273373 (±0.0034755545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 231.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.541.541.54
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z231.000231.000231.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0140.0360.000

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Supplemental data

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Mask #1

Fileemd_4631_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Tightly masked dimer component of alpha-1 antitrypsin polymers...

Fileemd_4631_additional.map
AnnotationTightly masked dimer component of alpha-1 antitrypsin polymers isolated from explant liver tissue; subunits rotated ~90 degrees around dimer axis.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Tightly masked dimer component of alpha-1 antitrypsin polymers...

Fileemd_4631_additional_1.map
AnnotationTightly masked dimer component of alpha-1 antitrypsin polymers isolated from explant liver tissue; subunits rotated ~90 degrees around dimer axis.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Polymers of the severe deficiency Z variant of alpha-1 antitrypsi...

EntireName: Polymers of the severe deficiency Z variant of alpha-1 antitrypsin labelled with 4B12 Fab fragments
Components
  • Tissue: Polymers of the severe deficiency Z variant of alpha-1 antitrypsin labelled with 4B12 Fab fragments
    • Complex: Complex between two 4B12 Fab fragments and a dimer component of polymers of the severe deficiency Z alpha-1 antitrypsin variant
      • Protein or peptide: Alpha-1 antitrypsin
      • Protein or peptide: 4B12 Fab heavy chain
      • Protein or peptide: 4B12 Fab light chain

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Supramolecule #1: Polymers of the severe deficiency Z variant of alpha-1 antitrypsi...

SupramoleculeName: Polymers of the severe deficiency Z variant of alpha-1 antitrypsin labelled with 4B12 Fab fragments
type: tissue / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Polymers of alpha-1 antitrypsin were isolated from patient explant tissue and labelled with the Fab fragment of a non-conformationally selective monoclonal antibody (4B12).
Source (natural)Organism: Homo sapiens (human) / Organ: Liver / Tissue: Explant tissue

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Supramolecule #2: Complex between two 4B12 Fab fragments and a dimer component of p...

SupramoleculeName: Complex between two 4B12 Fab fragments and a dimer component of polymers of the severe deficiency Z alpha-1 antitrypsin variant
type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human) / Organ: Liver
Molecular weightExperimental: 200 KDa

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Macromolecule #1: Alpha-1 antitrypsin

MacromoleculeName: Alpha-1 antitrypsin / type: protein_or_peptide / ID: 1
Details: 'Z' severe deficiency variant of alpha-1 antitrypsin
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Organ: Liver / Tissue: Explant tissue
SequenceString: EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFS LYR QLAHQSNSTN IFFSPVSIAT AFAMLSLGTK ADTHDEILEG LNFNLTEIPE AQIHEGF QE LLRTLNQPDS QLQLTTGNGL FLSEGLKLVD KFLEDVKKLY HSEAFTVNFG DTEEAKKQ I NDYVEKGTQG ...String:
EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFS LYR QLAHQSNSTN IFFSPVSIAT AFAMLSLGTK ADTHDEILEG LNFNLTEIPE AQIHEGF QE LLRTLNQPDS QLQLTTGNGL FLSEGLKLVD KFLEDVKKLY HSEAFTVNFG DTEEAKKQ I NDYVEKGTQG KIVDLVKELD RDTVFALVNY IFFKGKWERP FEVKDTEEED FHVDQVTTV KVPMMKRLGM FNIQHCKKLS SWVLLMKYLG NATAIFFLPD EGKLQHLENE LTHDIITKFL ENEDRRSAS LHLPKLSITG TYDLKSVLGQ LGITKVFSNG ADLSGVTEEA PLKLSKAVHK A VLTIDKKG TEAAGAMFLE AIPMSIPPEV KFNKPFVFLM IEQNTKSPLF MGKVVNPTQK

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Macromolecule #2: 4B12 Fab heavy chain

MacromoleculeName: 4B12 Fab heavy chain / type: protein_or_peptide / ID: 2
Details: Heavy chain of the Fab fragment of a non-conformationally-selective antibody (4B12) that binds to alpha-1-antitrypsin
Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: BALB/c / Organ: Spleen
SequenceString: QVKLEESGPE LVKPGASVKI SCKASGYSFI GYYMHWVKQS HVKSLEWIGR INPYNGATRY NQNFQDRATL TVDKSSSTAY MDFHSLTSED SAVYYCVRWP GDYWGQGTSV TVSSAKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV ...String:
QVKLEESGPE LVKPGASVKI SCKASGYSFI GYYMHWVKQS HVKSLEWIGR INPYNGATRY NQNFQDRATL TVDKSSSTAY MDFHSLTSED SAVYYCVRWP GDYWGQGTSV TVSSAKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRDCTS

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Macromolecule #3: 4B12 Fab light chain

MacromoleculeName: 4B12 Fab light chain / type: protein_or_peptide / ID: 3
Details: Light chain of the Fab fragment of a non-conformationally-selective antibody (4B12) that binds to alpha-1-antitrypsin
Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: BALB/c / Organ: Spleen
SequenceString: DIVMTQTPSS LSASLGGKVT ITCKASQDIN NYIAWYQLKP GKGPRQLIHY TSKLQPGIPS RFSGSGSGSD YSFSISNLEP EDIGTYYCLR YEDLWTFGGG TKLEIKRADA APTVSIFPPS SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS ...String:
DIVMTQTPSS LSASLGGKVT ITCKASQDIN NYIAWYQLKP GKGPRQLIHY TSKLQPGIPS RFSGSGSGSD YSFSISNLEP EDIGTYYCLR YEDLWTFGGG TKLEIKRADA APTVSIFPPS SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL TKDEYERHNS YTCEATHKTS TSPIVKSFN

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mM2-Amino-2-(hydroxymethyl)propane-1,3-diolTris
50.0 mMNaClSodium chloride
5.0 mMEDTA
StainingType: NEGATIVE / Material: Uranyl acetate
Details: 3 microlitres of prepared sample was applied to glow-discharged carbon film on 300 mesh copper (CF300-Cu) grids. Sample was bound to the grid for 1 minute before blotting excess. 5 ...Details: 3 microlitres of prepared sample was applied to glow-discharged carbon film on 300 mesh copper (CF300-Cu) grids. Sample was bound to the grid for 1 minute before blotting excess. 5 microlitres of 2% w/v uranyl acetate was applied for 1 minute before blotting; this staining step was repeated once.
GridMaterial: COPPER / Mesh: 300 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
DetailsThe sample comprised a mixed population of unbranched polymers of varying lengths, labelled with the Fab fragment of the 4B12 antibody.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Digitization - Dimensions - Width: 5120 pixel / Digitization - Dimensions - Height: 3840 pixel / Digitization - Sampling interval: 6.4 µm / Digitization - Frames/image: 2-30 / Number grids imaged: 1 / Number real images: 100 / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.009 µm / Calibrated defocus min: 0.888 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 41470
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 68538
Details: 8000 dimer particles were manually picked from the images, classified in RELION using Class2D, and 7 well-resolved 2D classes were used as autopicking references. Class2D was used with the ...Details: 8000 dimer particles were manually picked from the images, classified in RELION using Class2D, and 7 well-resolved 2D classes were used as autopicking references. Class2D was used with the output from autopicking to exclude classes containing 'junk' particles.
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 3)CTF parameters
Bsoft (ver. 1.8.9-20140223)Phase flipping (bctf -action 1)

Details: CTF correction was applied to the images prior to processing in RELION.
Startup modelType of model: OTHER
Details: A well-resolved 2D class showing a clear presence of Fab bound to alpha-1 antitrypsin was converted to a 3D reference with symmetric height/depth along the Z axis determined by pixel intensity.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 24.8 Å / Resolution method: FSC 0.33 CUT-OFF
Software:
Namedetails
RELION (ver. 2.1)Refine3D
RELION (ver. 2.1)PostProcess

Number images used: 6221
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1) / Software - details: Class3D
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1) / Software - details: Refine3D
Final 3D classificationNumber classes: 4 / Avg.num./class: 7744 / Software - Name: RELION (ver. 2.1) / Software - details: Class3D
Details: Successive rounds of 3D classification yielded two classes of dimers with well-resolved Fab protuberances, of which this database entry is one.
FSC plot (resolution estimation)

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