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- EMDB-43220: CryoEM structure of Angiopoietin-2 in complex with engineered con... -

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Basic information

Entry
Database: EMDB / ID: EMD-43220
TitleCryoEM structure of Angiopoietin-2 in complex with engineered conformationally rigid Fab 5A12.6DS
Map data
Sample
  • Complex: Angiopoietin-2 in complex with Fab 5A12.6DS
    • Complex: Angiopoietin-2
      • Protein or peptide: Angiopoietin-2
    • Complex: Fab 5A12.6DS
      • Protein or peptide: Fab 5A12.6DS heavy chain
      • Protein or peptide: Fab 5A12.6DS light chain
  • Ligand: CALCIUM ION
  • Ligand: water
Keywordsfab / antigen binding fragment / protein engineering / CYTOKINE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


negative regulation of positive chemotaxis / Tie signaling pathway / glomerulus vasculature development / positive regulation of coagulation / germ cell development / negative regulation of blood vessel endothelial cell migration / negative regulation of cell-substrate adhesion / animal organ regeneration / maternal process involved in female pregnancy / response to glucose ...negative regulation of positive chemotaxis / Tie signaling pathway / glomerulus vasculature development / positive regulation of coagulation / germ cell development / negative regulation of blood vessel endothelial cell migration / negative regulation of cell-substrate adhesion / animal organ regeneration / maternal process involved in female pregnancy / response to glucose / response to mechanical stimulus / Tie2 Signaling / negative regulation of angiogenesis / response to activity / cell projection / : / cellular response to growth factor stimulus / receptor tyrosine kinase binding / positive regulation of angiogenesis / blood coagulation / : / gene expression / angiogenesis / response to hypoxia / receptor ligand activity / signaling receptor binding / signal transduction / extracellular space / extracellular region / metal ion binding
Similarity search - Function
Fibrinogen alpha chain / Fibrinogen, conserved site / Fibrinogen C-terminal domain signature. / Fibrinogen-related domains (FReDs) / Fibrinogen beta and gamma chains, C-terminal globular domain / Fibrinogen, alpha/beta/gamma chain, C-terminal globular, subdomain 1 / Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain / Fibrinogen-like, C-terminal / Fibrinogen C-terminal domain profile.
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKung JE / Sudhamsu J
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: bioRxiv / Year: 2024
Title: Disulfi de constrained Fabs overcome target size limitation for high-resolution single-particle cryo-EM.
Authors: Jennifer E Kung / Matthew C Johnson / Christine C Jao / Christopher P Arthur / Dimitry Tegunov / Alexis Rohou / Jawahar Sudhamsu /
Abstract: High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure ...High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure determination of large proteins and their complexes, but a vast majority of proteins that underlie human diseases are small (< 50 kDa) and usually beyond its reach due to low signal-to-noise images and difficulties in particle alignment. Current strategies to overcome this problem increase the overall size of small protein targets using scaffold proteins that bind to the target, but are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the scaffolds. Here we present an iteratively engineered molecular design for transforming Fabs (antibody fragments), into conformationally rigid scaffolds (Rigid-Fabs) that, when bound to small proteins (~20 kDa), can enable high-resolution structure determination using cryo-EM. This design introduces multiple disulfide bonds at strategic locations, generates a well-folded Fab constrained into a rigid conformation and can be applied to Fabs from various species, isotypes and chimeric Fabs. We present examples of the Rigid Fab design enabling high-resolution (2.3-2.5 Å) structures of small proteins, Ang2 (26 kDa) and KRAS (21 kDa) by cryo-EM. The strategies for designing disulfide constrained Rigid Fabs in our work thus establish a general approach to overcome the target size limitation of single particle cryo-EM.
History
DepositionDec 27, 2023-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43220.png

Downloads & links

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Map

FileDownload / File: emd_43220.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 324 pix.
= 335.21 Å		1.03 Å/pix.
x 324 pix.
= 335.21 Å		1.03 Å/pix.
x 324 pix.
= 335.21 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0346 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.19
Minimum - Maximum-0.40889502 - 0.91151774
Average (Standard dev.)-0.00053077145 (±0.011496608)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 335.21042 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43220_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_43220_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Angiopoietin-2 in complex with Fab 5A12.6DS

EntireName: Angiopoietin-2 in complex with Fab 5A12.6DS
Components
  • Complex: Angiopoietin-2 in complex with Fab 5A12.6DS
    • Complex: Angiopoietin-2
      • Protein or peptide: Angiopoietin-2
    • Complex: Fab 5A12.6DS
      • Protein or peptide: Fab 5A12.6DS heavy chain
      • Protein or peptide: Fab 5A12.6DS light chain
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: Angiopoietin-2 in complex with Fab 5A12.6DS

SupramoleculeName: Angiopoietin-2 in complex with Fab 5A12.6DS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: Angiopoietin-2

SupramoleculeName: Angiopoietin-2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Fab 5A12.6DS

SupramoleculeName: Fab 5A12.6DS / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Angiopoietin-2

MacromoleculeName: Angiopoietin-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.530371 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: AGSEQISFRD CAEVFKSGHT TNGIYTLTFP NSTEEIKAYC DMEAGGGGWT IIQRREDGSV DFQRTWKEYK VGFGNPSGEY WLGNEFVSQ LTNQQRYVLK IHLKDWEGNE AYSLYEHFYL SSEELNYRIH LKGLTGTAGK ISSISQPGND FSTKDGDNDK C ICKCSQML ...String:
AGSEQISFRD CAEVFKSGHT TNGIYTLTFP NSTEEIKAYC DMEAGGGGWT IIQRREDGSV DFQRTWKEYK VGFGNPSGEY WLGNEFVSQ LTNQQRYVLK IHLKDWEGNE AYSLYEHFYL SSEELNYRIH LKGLTGTAGK ISSISQPGND FSTKDGDNDK C ICKCSQML TGGWWFDACG PSNLNGMYYP QRQNTNKFNG IKWYYWKGSG YSLKATTMMI RPADFGNSHH HHHH

UniProtKB: Angiopoietin-2

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Macromolecule #2: Fab 5A12.6DS heavy chain

MacromoleculeName: Fab 5A12.6DS heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.234367 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG CVQPGGSLRL SCAASGFTIS DYWIHWVRQA PGKGLEWVAG ITPAGGYTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARF VFFLPYAMDY WGQGTCVTVS SASTKGPSVC PLAPSSKSTS GGTACLGCLV KDYFCECPVT V SWNSGALT ...String:
EVQLVESGGG CVQPGGSLRL SCAASGFTIS DYWIHWVRQA PGKGLEWVAG ITPAGGYTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARF VFFLPYAMDY WGQGTCVTVS SASTKGPSVC PLAPSSKSTS GGTACLGCLV KDYFCECPVT V SWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH THHHHHHP

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Macromolecule #3: Fab 5A12.6DS light chain

MacromoleculeName: Fab 5A12.6DS light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.23007 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQFLS SFGVAWYQQK CGKAPKLLIY GASSLYSGVP SRFSGSGSGT DFTLTISSLQ CEDFATYYC QQGLLSPLTF GQGTKVEIKR TVAAPSVCIF PPSDECLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTCQD ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQFLS SFGVAWYQQK CGKAPKLLIY GASSLYSGVP SRFSGSGSGT DFTLTISSLQ CEDFATYYC QQGLLSPLTF GQGTKVEIKR TVAAPSVCIF PPSDECLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTCQD SKDCTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 43 / 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

BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMsodium chlorideNaCl
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Details: The peak fraction was subjected to mild crosslinking with 0.5 mM BS3 at room temperature for 10 min. The crosslinking reaction was quenched by addition of 100mM Tris pH 7.5.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 68.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination 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: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 1017611
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelPDB ID:

4zfg


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8vgp:
CryoEM structure of Angiopoietin-2 in complex with engineered conformationally rigid Fab 5A12.6DS

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