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- EMDB-43203: CryoEM structure of tryptase in complex with engineered conformat... -

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Basic information

Entry
Database: EMDB / ID: EMD-43203
TitleCryoEM structure of tryptase in complex with engineered conformationally rigid anti-tryptase Fab E104.v1.6DS
Map data
Sample
  • Complex: Tryptase tetramer in complex with Fab E104.v1.6DS
    • Complex: Tryptase tetramer
      • Protein or peptide: Tryptase alpha/beta-1
    • Complex: anti-tryptase Fab E104.v1.6DS
      • Protein or peptide: Fab E104.v1.6DS light chain
      • Protein or peptide: Fab E104.v1.6DS light chain
  • Ligand: water
Keywordsantibody 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsKung JE / Johnson MC / 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_43203.png

Downloads & links

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Map

FileDownload / File: emd_43203.map.gz / Format: CCP4 / Size: 244.1 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 Å/pix.
x 400 pix.
= 400. Å		1 Å/pix.
x 400 pix.
= 400. Å		1 Å/pix.
x 400 pix.
= 400. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.4
Minimum - Maximum-6.2734394 - 18.161477999999999
Average (Standard dev.)-0.007634084 (±0.35003424)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 400.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43203_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43203_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 : Tryptase tetramer in complex with Fab E104.v1.6DS

EntireName: Tryptase tetramer in complex with Fab E104.v1.6DS
Components
  • Complex: Tryptase tetramer in complex with Fab E104.v1.6DS
    • Complex: Tryptase tetramer
      • Protein or peptide: Tryptase alpha/beta-1
    • Complex: anti-tryptase Fab E104.v1.6DS
      • Protein or peptide: Fab E104.v1.6DS light chain
      • Protein or peptide: Fab E104.v1.6DS light chain
  • Ligand: water

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Supramolecule #1: Tryptase tetramer in complex with Fab E104.v1.6DS

SupramoleculeName: Tryptase tetramer in complex with Fab E104.v1.6DS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: Tryptase tetramer

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

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Supramolecule #3: anti-tryptase Fab E104.v1.6DS

SupramoleculeName: anti-tryptase Fab E104.v1.6DS / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Tryptase alpha/beta-1

MacromoleculeName: Tryptase alpha/beta-1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: tryptase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.476348 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: IVGGQEAPRS KWPWQVSLRV HGPYWMHFCG GSLIHPQWVL TAAHCVGPDV KDLAALRVQL REQHLYYQDQ LLPVSRIIVH PQFYTAQIG ADIALLELEE PVNVSSHVHT VTLPPASETF PPGMPCWVTG WGDVDNDERL PPPFPLKQVK VPIMENHICD A KYHLGAYT ...String:
IVGGQEAPRS KWPWQVSLRV HGPYWMHFCG GSLIHPQWVL TAAHCVGPDV KDLAALRVQL REQHLYYQDQ LLPVSRIIVH PQFYTAQIG ADIALLELEE PVNVSSHVHT VTLPPASETF PPGMPCWVTG WGDVDNDERL PPPFPLKQVK VPIMENHICD A KYHLGAYT GDDVRIVRDD MLCAGNTRRD SCQGDSGGPL VCKVNGTWLQ AGVVSWGEGC AQPNRPGIYT RVTYYLDWIH HY VPKKP

UniProtKB: Tryptase alpha/beta-1

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Macromolecule #2: Fab E104.v1.6DS light chain

MacromoleculeName: Fab E104.v1.6DS light chain / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.534291 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCQSIKSVY NNRLGWYQQK CGKAPKLLIY ETSILTSGVP SRFSGSGSGT DFTLTISSLQ CEDFATYYC AGGFDRSGDT TFGQGTKVEI KRTVAAPSVC IFPPSDECLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTC ...String:
DIQMTQSPSS LSASVGDRVT ITCQSIKSVY NNRLGWYQQK CGKAPKLLIY ETSILTSGVP SRFSGSGSGT DFTLTISSLQ CEDFATYYC AGGFDRSGDT TFGQGTKVEI KRTVAAPSVC IFPPSDECLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTC QDSKDCTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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Macromolecule #3: Fab E104.v1.6DS light chain

MacromoleculeName: Fab E104.v1.6DS light chain / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.382652 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGPG CVKPSETLSL TCTVSRFSLI GYAITWIRQP PGKGLEWIGG ISSAATTFYS SWAKSRVTIS VDTSKNQFSL KLSSVTAAD TAVYYCARDP RGYGAALDRL DLWGQGTCVT VSSFASTKGP SVCPLAPSSK STSGGTACLG CLVKDYFCEC P VTVSWNSG ...String:
EVQLVESGPG CVKPSETLSL TCTVSRFSLI GYAITWIRQP PGKGLEWIGG ISSAATTFYS SWAKSRVTIS VDTSKNQFSL KLSSVTAAD TAVYYCARDP RGYGAALDRL DLWGQGTCVT VSSFASTKGP SVCPLAPSSK STSGGTACLG CLVKDYFCEC P VTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTHHHHHH P

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 135 / 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: 5.5
Component:
ConcentrationNameFormula
20.0 mMMOPS
800.0 mMsodium chlorideNaCl
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
Details: Grid was treated overnight with 4 mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol then rinsed in ethanol prior to sample application.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Average electron dose: 64.0 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: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 253508
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cisTEM

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Atomic model buiding 1

Initial modelPDB ID:

6vvu


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8vgk:
CryoEM structure of tryptase in complex with engineered conformationally rigid anti-tryptase Fab E104.v1.6DS

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