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Yorodumi- EMDB-43203: CryoEM structure of tryptase in complex with engineered conformat... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-43203 | |||||||||
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Title | CryoEM structure of tryptase in complex with engineered conformationally rigid anti-tryptase Fab E104.v1.6DS | |||||||||
Map data | ||||||||||
Sample |
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Keywords | 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 | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.4 Å | |||||||||
Authors | Kung JE / Johnson MC / Sudhamsu J | |||||||||
Funding support | 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_43203.map.gz | 226.2 MB | EMDB map data format | |
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Header (meta data) | emd-43203-v30.xml emd-43203.xml | 19.3 KB 19.3 KB | Display Display | EMDB header |
Images | emd_43203.png | 40.4 KB | ||
Filedesc metadata | emd-43203.cif.gz | 6.5 KB | ||
Others | emd_43203_half_map_1.map.gz emd_43203_half_map_2.map.gz | 29.6 MB 29.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-43203 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-43203 | HTTPS FTP |
-Validation report
Summary document | emd_43203_validation.pdf.gz | 663.9 KB | Display | EMDB validaton report |
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Full document | emd_43203_full_validation.pdf.gz | 663.5 KB | Display | |
Data in XML | emd_43203_validation.xml.gz | 13.8 KB | Display | |
Data in CIF | emd_43203_validation.cif.gz | 16.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43203 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43203 | HTTPS FTP |
-Related structure data
Related structure data | 8vgkMC 8vgeC 8vgfC 8vggC 8vghC 8vgiC 8vgjC 8vglC 8vgmC 8vgpC 8vgqC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_43203.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_43203_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_43203_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Tryptase tetramer in complex with Fab E104.v1.6DS
Entire | Name: Tryptase tetramer in complex with Fab E104.v1.6DS |
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Components |
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-Supramolecule #1: Tryptase tetramer in complex with Fab E104.v1.6DS
Supramolecule | Name: 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
Supramolecule | Name: Tryptase tetramer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) |
-Supramolecule #3: anti-tryptase Fab E104.v1.6DS
Supramolecule | Name: anti-tryptase Fab E104.v1.6DS / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 |
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Source (natural) | Organism: Homo sapiens (human) |
-Macromolecule #1: Tryptase alpha/beta-1
Macromolecule | Name: Tryptase alpha/beta-1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: tryptase |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 27.476348 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: 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 |
-Macromolecule #2: Fab E104.v1.6DS light chain
Macromolecule | Name: Fab E104.v1.6DS light chain / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 23.534291 KDa |
Recombinant expression | Organism: Cricetulus griseus (Chinese hamster) |
Sequence | String: 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 |
-Macromolecule #3: Fab E104.v1.6DS light chain
Macromolecule | Name: Fab E104.v1.6DS light chain / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 25.382652 KDa |
Recombinant expression | Organism: Cricetulus griseus (Chinese hamster) |
Sequence | String: 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 |
-Macromolecule #4: water
Macromolecule | Name: water / type: ligand / ID: 4 / Number of copies: 135 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 5.5 Component:
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Grid | Model: 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. | |||||||||
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Average electron dose: 64.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 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 |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Applied 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 assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC |
Final angle assignment | Type: OTHER / Software - Name: cisTEM |