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- PDB-9dvq: Cryo-EM structure of Human Fibroblast Activation Protein alpha di... -

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Basic information

Entry
Database: PDB / ID: 9dvq
TitleCryo-EM structure of Human Fibroblast Activation Protein alpha dimer with one SUMO-I3 VHHs bound
Components
  • Antiplasmin-cleaving enzyme FAP, soluble form
  • SUMO-I3
KeywordsHYDROLASE / Fibroblast activation protein / single domain antibody / Protease
Function / homology
Function and homology information


negative regulation of extracellular matrix organization / melanocyte proliferation / peptidase complex / melanocyte apoptotic process / regulation of collagen catabolic process / negative regulation of cell proliferation involved in contact inhibition / prolyl oligopeptidase / negative regulation of extracellular matrix disassembly / dipeptidyl-peptidase IV / basal part of cell ...negative regulation of extracellular matrix organization / melanocyte proliferation / peptidase complex / melanocyte apoptotic process / regulation of collagen catabolic process / negative regulation of cell proliferation involved in contact inhibition / prolyl oligopeptidase / negative regulation of extracellular matrix disassembly / dipeptidyl-peptidase IV / basal part of cell / regulation of fibrinolysis / dipeptidyl-peptidase activity / lamellipodium membrane / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / positive regulation of execution phase of apoptosis / endothelial cell migration / serine-type peptidase activity / proteolysis involved in protein catabolic process / ruffle membrane / integrin binding / apical part of cell / peptidase activity / lamellipodium / protease binding / angiogenesis / endopeptidase activity / regulation of cell cycle / cell adhesion / serine-type endopeptidase activity / focal adhesion / cell surface / protein homodimerization activity / proteolysis / extracellular space / identical protein binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
: / Peptidase S9, serine active site / Dipeptidylpeptidase IV, N-terminal domain / Dipeptidyl peptidase IV (DPP IV) N-terminal region / Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Prolyl endopeptidase FAP
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsXu, Z. / Schnicker, N.J. / Wadas, T.J.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R21-CA219899 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R21-CA227709 United States
Department of Defense (DOD, United States)W81XMH-19-1-0046 United States
CitationJournal: RSC Chem Biol / Year: 2025
Title: Cryo-electron microscopy reveals a single domain antibody with a unique binding epitope on fibroblast activation protein alpha.
Authors: Zhen Xu / Akesh Sinha / Darpan N Pandya / Nicholas J Schnicker / Thaddeus J Wadas /
Abstract: Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this ...Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this unique expression profile, designing functional diagnostic and therapeutic agents that effectively target this biomarker remains elusive. Here we report the structural characterization of the interaction between a novel single domain antibody (sdAb), I3, and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 Å and contained two distinct populations; one I3 bound and two I3 molecules bound to the FAP dimer. In both cases, the sdAb bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this report describes the rational mutation of specific residues within the complementarity determining region 3 (CDR3) loop to enhance affinity and selectivity of the I3 molecule for FAP. This report represents the first sdAb-FAP structure to be described in the literature.
History
DepositionOct 8, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 26, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Antiplasmin-cleaving enzyme FAP, soluble form
B: Antiplasmin-cleaving enzyme FAP, soluble form
G: SUMO-I3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,24011
Polymers199,4713
Non-polymers1,7708
Water00
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 Antiplasmin-cleaving enzyme FAP, soluble form / APCE


Mass: 86516.211 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FAP / Production host: Cricetulus griseus (Chinese hamster)
References: UniProt: Q12884, dipeptidyl-peptidase IV, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases, prolyl oligopeptidase
#2: Antibody SUMO-I3


Mass: 26438.318 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
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
1Complex of Human Fibroblast activation protein alpha with SUMO-I3COMPLEX#1-#20MULTIPLE SOURCES
2Human Fibroblast activation protein alphaCOMPLEX#11RECOMBINANT
3SUMO-I3COMPLEX#21RECOMBINANT
Molecular weightValue: 0.23 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Cricetulus griseus (Chinese hamster)10029
33Escherichia coli (E. coli)562
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 5450
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Category: model refinement
Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
12.7FSC 0.143 CUT-OFF27271119DVQPOINT
22.7FSC 0.143 CUT-OFF27271119DVQPOINT
32.7FSC 0.143 CUT-OFF27271129DVQPOINT
42.7FSC 0.143 CUT-OFF27271129DVQPOINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11Z68

1z68

11Z681PDBexperimental model
21AlphaFoldin silico model
RefinementHighest resolution: 2.7 Å

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