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- EMDB-71532: Angiopoietin-2 in complex with Fab 5A12.WT, used for comparison w... -

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Basic information

Entry
Database: EMDB / ID: EMD-71532
TitleAngiopoietin-2 in complex with Fab 5A12.WT, used for comparison with Fab 5A12.6DS
Map data
Sample
  • Complex: Ang2:WT Fab
    • Protein or peptide: Angiopoietin-2
    • Protein or peptide: Fab 5A12.WT heavy chain
    • Protein or peptide: Fab 5A12.WT light chain
Keywordsfab / antigen binding fragment / protein engineering / CYTOKINE-IMMUNE SYSTEM complex / CYTOKINE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsKung J / Johnson MC / Tegunov D / Jao CC / Wu P / Oh A / Lin M / Daria JM / Koth CM / Arthur CP ...Kung J / Johnson MC / Tegunov D / Jao CC / Wu P / Oh A / Lin M / Daria JM / Koth CM / Arthur CP / Rohou A / Sudhamsu J
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2025
Title: Disulfide-constrained Fabs overcome target size limitation for high-resolution single particle cryoEM.
Authors: Jennifer E Kung / Matthew C Johnson / Dimitry Tegunov / Christine C Jao / Ping Wu / Angela Oh / May Lin / Jose M Daria / Christopher M Koth / Christopher P Arthur / Alexis Rohou / Jawahar Sudhamsu /
Abstract: High-resolution protein structures are essential for understanding biological mechanisms and drug discovery. While cryoEM has revolutionized structure determination of large protein complexes, most ...High-resolution protein structures are essential for understanding biological mechanisms and drug discovery. While cryoEM has revolutionized structure determination of large protein complexes, most disease-related proteins are small (<50 kDa) and challenging to resolve due to low signal-to-noise ratios and alignment difficulties. Current scaffold protein strategies increase target size but suffer from inherent flexibility, resulting in poorly resolved targets compared to scaffolds. We present an iteratively engineered molecular design transforming antibody fragments (Fabs) into conformationally Rigid Fabs that enable high-resolution structure determination of small proteins (~20 kDa). This design introduces strategic disulfide bonds, creating well-folded, rigidly constrained Fabs applicable across various species, frameworks, and chimeric constructs. Rigid Fabs enabled high-resolution cryoEM structures (2.3-2.5 Å) of two small proteins: Ang2 (26 kDa) and KRAS (21 kDa). Our disulfide-constrained Rigid Fab strategy provides a general approach for overcoming target size limitation of single-particle cryoEM.
History
DepositionJun 30, 2025-
Header (metadata) releaseOct 15, 2025-
Map releaseOct 15, 2025-
UpdateOct 15, 2025-
Current statusOct 15, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71532.png

Downloads & links

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Map

FileDownload / File: emd_71532.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 360 pix.
= 263.16 Å		0.73 Å/pix.
x 360 pix.
= 263.16 Å		0.73 Å/pix.
x 360 pix.
= 263.16 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.731 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.095
Minimum - Maximum-0.37109375 - 0.64453125
Average (Standard dev.)0.00028550756 (±0.008569297)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 263.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_71532_msk_1.map
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Additional map: #1

Fileemd_71532_additional_1.map
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Half map: #2

Fileemd_71532_half_map_1.map
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Half map: #1

Fileemd_71532_half_map_2.map
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Sample components

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Entire : Ang2:WT Fab

EntireName: Ang2:WT Fab
Components
  • Complex: Ang2:WT Fab
    • Protein or peptide: Angiopoietin-2
    • Protein or peptide: Fab 5A12.WT heavy chain
    • Protein or peptide: Fab 5A12.WT light chain

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Supramolecule #1: Ang2:WT Fab

SupramoleculeName: Ang2:WT Fab / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 74.8 KDa

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Macromolecule #1: Angiopoietin-2

MacromoleculeName: Angiopoietin-2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: AGSEQISFRD CAEVFKSGHT TNGIYTLTFP NSTEEIKAYC DMEAGGGGWT IIQRREDGSV DFQRTWKEYK VGFGNPSGEY WLGNEFVSQL TNQQRYVLKI HLKDWEGNEA YSLYEHFYLS SEELNYRIHL KGLTGTAGKI SSISQPGNDF STKDGDNDKC ICKCSQMLTG ...String:
AGSEQISFRD CAEVFKSGHT TNGIYTLTFP NSTEEIKAYC DMEAGGGGWT IIQRREDGSV DFQRTWKEYK VGFGNPSGEY WLGNEFVSQL TNQQRYVLKI HLKDWEGNEA YSLYEHFYLS SEELNYRIHL KGLTGTAGKI SSISQPGNDF STKDGDNDKC ICKCSQMLTG GWWFDACGPS NLNGMYYPQR QNTNKFNGIK WYYWKGSGYS LKATTMMIRP ADFGNSHHHH HH

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Macromolecule #2: Fab 5A12.WT heavy chain

MacromoleculeName: Fab 5A12.WT heavy chain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFTIS DYWIHWVRQA PGKGLEWVAG ITPAGGYTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARFV FFLPYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFTIS DYWIHWVRQA PGKGLEWVAG ITPAGGYTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARFV FFLPYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTHH HHHHP

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Macromolecule #3: Fab 5A12.WT light chain

MacromoleculeName: Fab 5A12.WT light chain / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQFLS SFGVAWYQQK PGKAPKLLIY GASSLYSGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QGLLSPLTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQFLS SFGVAWYQQK PGKAPKLLIY GASSLYSGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QGLLSPLTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 10560 / Average electron dose: 45.0 e/Å2
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 / Nominal magnification: 165000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 20000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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