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- PDB-8vgl: CryoEM structure of Nav1.7 in complex with wild type Fab 7A9 -

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Basic information

Entry
Database: PDB / ID: 8vgl
TitleCryoEM structure of Nav1.7 in complex with wild type Fab 7A9
Components
  • Chimeric Nav1.7-NavAb
  • Fab 7A9 heavy chain
  • Fab 7A9 light chain
KeywordsTRANSPORT PROTEIN / antibody fragment / fab / protein engineering / ion channel
Biological speciesAliarcobacter butzleri RM4018 (bacteria)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsKung, J.E. / Jao, C.C. / Arthur, C.P. / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chimeric Nav1.7-NavAb
B: Chimeric Nav1.7-NavAb
C: Chimeric Nav1.7-NavAb
D: Chimeric Nav1.7-NavAb
H: Fab 7A9 heavy chain
L: Fab 7A9 light chain
F: Fab 7A9 light chain
E: Fab 7A9 heavy chain


Theoretical massNumber of molelcules
Total (without water)234,4878
Polymers234,4878
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Chimeric Nav1.7-NavAb


Mass: 34618.094 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aliarcobacter butzleri RM4018 (bacteria)
Production host: Trichoplusia ni (cabbage looper)
#2: Antibody Fab 7A9 heavy chain


Mass: 24523.518 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody Fab 7A9 light chain


Mass: 23483.910 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
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
1Nav1.7 in complex with Fab 7A9COMPLEXall0RECOMBINANT
2Nav1.7COMPLEX#11RECOMBINANT
3anti-Nav1.7 Fab 7A9COMPLEX#2-#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Aliarcobacter butzleri RM4018 (bacteria)367737
32Aliarcobacter butzleri RM4018 (bacteria)367737
43Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trichoplusia ni (cabbage looper)7111
32Trichoplusia ni (cabbage looper)7111
43Cricetulus griseus (Chinese hamster)10029
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris1
2100 mMsodium chlorideNaCl1
30.042 %GDN1
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 R2/2
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: 165000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 44 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
8ISOLDEmodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
14PHENIX1.21rc1_5049model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 391335 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16N4Q

6n4q

16N4Q1PDBexperimental model
25EK0

5ek0

15EK02PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316008
ELECTRON MICROSCOPYf_angle_d0.54121762
ELECTRON MICROSCOPYf_dihedral_angle_d13.6525708
ELECTRON MICROSCOPYf_chiral_restr0.042530
ELECTRON MICROSCOPYf_plane_restr0.0092678

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