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- PDB-8vgk: CryoEM structure of tryptase in complex with engineered conformat... -

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Basic information

Entry
Database: PDB / ID: 8vgk
TitleCryoEM structure of tryptase in complex with engineered conformationally rigid anti-tryptase Fab E104.v1.6DS
Components
  • (Fab E104.v1.6DS light chain) x 2
  • Tryptase alpha/beta-1
KeywordsHYDROLASE/IMMUNE SYSTEM / antibody fragment / fab / protein engineering / tryptase / HYDROLASE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


tryptase / Activation of Matrix Metalloproteinases / extracellular matrix disassembly / serine-type peptidase activity / defense response / serine-type endopeptidase activity / proteolysis / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Serine proteases, trypsin family, histidine active site. / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Tryptase alpha/beta-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsKung, J.E. / Johnson, M.C. / 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: Tryptase alpha/beta-1
B: Tryptase alpha/beta-1
C: Tryptase alpha/beta-1
D: Tryptase alpha/beta-1
E: Fab E104.v1.6DS light chain
F: Fab E104.v1.6DS light chain
G: Fab E104.v1.6DS light chain
H: Fab E104.v1.6DS light chain
I: Fab E104.v1.6DS light chain
J: Fab E104.v1.6DS light chain
K: Fab E104.v1.6DS light chain
L: Fab E104.v1.6DS light chain


Theoretical massNumber of molelcules
Total (without water)305,57312
Polymers305,57312
Non-polymers00
Water2,432135
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
Tryptase alpha/beta-1 / Tryptase-1 / Tryptase I / Tryptase alpha-1


Mass: 27476.348 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TPSAB1, TPS1, TPS2, TPSB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15661, tryptase
#2: Antibody
Fab E104.v1.6DS light chain


Mass: 23534.291 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody
Fab E104.v1.6DS light chain


Mass: 25382.652 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 135 / Source method: isolated from a natural source / Formula: H2O
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
1Tryptase tetramer in complex with Fab E104.v1.6DSCOMPLEX#1-#30MULTIPLE SOURCES
2Tryptase tetramerCOMPLEX#11RECOMBINANT
3anti-tryptase Fab E104.v1.6DSCOMPLEX#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: 5.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMMOPS1
2800 mMsodium chlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grid was treated overnight with 4 mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol then rinsed in ethanol prior to sample application.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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: 64 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cisTEMparticle selection
2SerialEMimage acquisition
4cisTEMCTF correction
7UCSF ChimeraXmodel fitting
8ISOLDEmodel fitting
10cryoSPARCinitial Euler assignment
11cisTEMfinal Euler assignment
13cisTEM3D reconstruction
20PHENIX1.21rc1_5049model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 253508 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6VVU

6vvu


Accession code: 6VVU / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00520960
ELECTRON MICROSCOPYf_angle_d1.03828592
ELECTRON MICROSCOPYf_dihedral_angle_d13.8417480
ELECTRON MICROSCOPYf_chiral_restr0.063220
ELECTRON MICROSCOPYf_plane_restr0.013648

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