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- EMDB-43211: Constituent map: Focused refinement of Nav1.7 in complex of Nav1.... -

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Basic information

Entry
Database: EMDB / ID: EMD-43211
TitleConstituent map: Focused refinement of Nav1.7 in complex of Nav1.7 and Fab 7A9.4DS
Map data
Sample
  • Complex: Nav1.7 in complex with Fab 7A9.4DS
    • Complex: Nav1.7
      • Protein or peptide: Chimeric Nav1.7/NavAb
    • Complex: anti-Nav1.7 Fab 7A9.4DS
      • Protein or peptide: Fab 7A9.4DS heavy chain
      • Protein or peptide: Fab 7A9.4DS light chain
Keywordsantibody fragment / fab / protein engineering / ion channel / TRANSPORT PROTEIN
Biological speciesAliarcobacter butzleri RM4018 (bacteria) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsKung JE / Jao CC / Arthur CP / 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-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43211.png

Downloads & links

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Map

FileDownload / File: emd_43211.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.)
0.94 Å/pix.
x 400 pix.
= 374.28 Å		0.94 Å/pix.
x 400 pix.
= 374.28 Å		0.94 Å/pix.
x 400 pix.
= 374.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9357 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.4625543 - 0.88778144
Average (Standard dev.)-0.00038126475 (±0.012914462)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 374.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43211_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_43211_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_43211_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 : Nav1.7 in complex with Fab 7A9.4DS

EntireName: Nav1.7 in complex with Fab 7A9.4DS
Components
  • Complex: Nav1.7 in complex with Fab 7A9.4DS
    • Complex: Nav1.7
      • Protein or peptide: Chimeric Nav1.7/NavAb
    • Complex: anti-Nav1.7 Fab 7A9.4DS
      • Protein or peptide: Fab 7A9.4DS heavy chain
      • Protein or peptide: Fab 7A9.4DS light chain

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Supramolecule #1: Nav1.7 in complex with Fab 7A9.4DS

SupramoleculeName: Nav1.7 in complex with Fab 7A9.4DS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Aliarcobacter butzleri RM4018 (bacteria)

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Supramolecule #2: Nav1.7

SupramoleculeName: Nav1.7 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Aliarcobacter butzleri RM4018 (bacteria)

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Supramolecule #3: anti-Nav1.7 Fab 7A9.4DS

SupramoleculeName: anti-Nav1.7 Fab 7A9.4DS / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Chimeric Nav1.7/NavAb

MacromoleculeName: Chimeric Nav1.7/NavAb / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Aliarcobacter butzleri RM4018 (bacteria)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: PRGSHMYLRI TNIVESSFFT KFIIYLIVLN MVTMMVEKEG QSQHMTEVLY WINVVFIILF TIEIILRIYV HRISFFKDPW SLFDFVVVI ISIVGMFLAD LIETYFVSPT LFRVIRLARI GRILRLVTAV PQMRKIVSAL ISVIPGMLSV IALMTLFFYI F AIMATQLF ...String:
PRGSHMYLRI TNIVESSFFT KFIIYLIVLN MVTMMVEKEG QSQHMTEVLY WINVVFIILF TIEIILRIYV HRISFFKDPW SLFDFVVVI ISIVGMFLAD LIETYFVSPT LFRVIRLARI GRILRLVTAV PQMRKIVSAL ISVIPGMLSV IALMTLFFYI F AIMATQLF GERFPEWFGT LGESFYTLFQ VMTLESWSMG IVRPLMEVYP YAWVFFIPFI FVVTFVMINL VVAIIVDAMA IL NQKEEQH IIDEVQSHED NINNEIIKLR EEIVELKELI KTSLKN

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Macromolecule #2: Fab 7A9.4DS heavy chain

MacromoleculeName: Fab 7A9.4DS heavy chain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG CVKPGGSLKL SCAASGFTFS NYAMSWVRQT PEKRLEWVAT ISNGGRYTYY PDSVKGRFTI SRDNAKNSLY LQMSSLRSED TAMYYCARHL YRYDVGGALD YWGQGTCVTV SSAKTTAPSV YPLAPVCGDT TGSSVTLGCL VKGYFCECPV TLTWNSGSLS ...String:
EVQLVESGGG CVKPGGSLKL SCAASGFTFS NYAMSWVRQT PEKRLEWVAT ISNGGRYTYY PDSVKGRFTI SRDNAKNSLY LQMSSLRSED TAMYYCARHL YRYDVGGALD YWGQGTCVTV SSAKTTAPSV YPLAPVCGDT TGSSVTLGCL VKGYFCECPV TLTWNSGSLS SGVHTFPAVL QSDLYTLSSS VTVTSSTWPS QSITCNVAHP ASSTKVDKKI EPRGPTIKPH HHHHHP

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Macromolecule #3: Fab 7A9.4DS light chain

MacromoleculeName: Fab 7A9.4DS light chain / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EIVLTQSPAL MAASPGEKVT ITCSVSLSIS SSNLFWYQQK CETSPKPWIY GTSKLASGVP VRFSGSGSGT SYSLTISSME CEDAATYYCQ QWSSHSFTFG GGTKLEIKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV LNSWTCQDSK ...String:
EIVLTQSPAL MAASPGEKVT ITCSVSLSIS SSNLFWYQQK CETSPKPWIY GTSKLASGVP VRFSGSGSGT SYSLTISSME CEDAATYYCQ QWSSHSFTFG GGTKLEIKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV LNSWTCQDSK DCTYSMSSTL TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC

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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: 8
Component:
ConcentrationNameFormula
10.0 mMTris
100.0 mMsodium chlorideNaCl
0.042 %GDN
GridModel: Quantifoil R2/2 / 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: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Average electron dose: 44.0 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: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
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: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 362778
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial model
PDB IDChain

6n4q

source_name: PDB, initial_model_type: experimental model

5ek0

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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