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- PDB-8vgp: CryoEM structure of Angiopoietin-2 in complex with engineered con... -

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Basic information

Entry
Database: PDB / ID: 8vgp
TitleCryoEM structure of Angiopoietin-2 in complex with engineered conformationally rigid Fab 5A12.6DS
Components
  • Angiopoietin-2
  • Fab 5A12.6DS heavy chain
  • Fab 5A12.6DS light chain
KeywordsCYTOKINE/IMMUNE SYSTEM / fab / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKung, J.E. / Sudhamsu, J.
Funding support1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Angiopoietin-2
H: Fab 5A12.6DS heavy chain
L: Fab 5A12.6DS light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,0354
Polymers74,9953
Non-polymers401
Water77543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Angiopoietin-2 / ANG-2


Mass: 26530.371 Da / Num. of mol.: 1 / Fragment: Receptor binding domain, UNP residues 225-444
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANGPT2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O15123
#2: Antibody Fab 5A12.6DS heavy chain


Mass: 25234.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody Fab 5A12.6DS light chain


Mass: 23230.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 43 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Angiopoietin-2 in complex with Fab 5A12.6DSCOMPLEX#1-#30MULTIPLE SOURCES
2Angiopoietin-2COMPLEX#11RECOMBINANT
3Fab 5A12.6DSCOMPLEX#2-#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
32Trichoplusia ni (cabbage looper)7111
43Cricetulus griseus (Chinese hamster)10029
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMsodium chlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 68.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7ISOLDEmodel fitting
8UCSF ChimeraXmodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
14PHENIX1.21rc1_5049model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1017611 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 4ZFG

4zfg


Accession code: 4ZFG / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.015128
ELECTRON MICROSCOPYf_angle_d1.0176959
ELECTRON MICROSCOPYf_dihedral_angle_d14.3411818
ELECTRON MICROSCOPYf_chiral_restr0.063750
ELECTRON MICROSCOPYf_plane_restr0.021890

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