[English] 日本語
Yorodumi
- EMDB-43204: Composite cryoEM map of Nav1.7 in complex with wild type Fab 7A9 -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-43204
TitleComposite cryoEM map of Nav1.7 in complex with wild type Fab 7A9
Map data
Sample
  • Complex: Nav1.7 in complex with Fab 7A9
    • Complex: Nav1.7
      • Protein or peptide: Chimeric Nav1.7-NavAb
    • Complex: anti-Nav1.7 Fab 7A9
      • Protein or peptide: Fab 7A9 heavy chain
      • Protein or peptide: Fab 7A9 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: 2.6 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

-
Structure visualization

Supplemental images

emd_43204.png

Downloads & links

-
Map

FileDownload / File: emd_43204.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
AxesX (Sec.)Y (Row.)Z (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: 7.1
Minimum - Maximum-25.856864999999999 - 61.519145999999999
Average (Standard dev.)-0.0000010320093 (±0.9999832)
SymmetrySpace group: 1
Details

EMDB XML:

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

-
Supplemental data

-
Half map: #2

Fileemd_43204_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #1

Fileemd_43204_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : Nav1.7 in complex with Fab 7A9

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

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

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

-
Supramolecule #2: Nav1.7

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

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

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

-
Macromolecule #1: Chimeric Nav1.7-NavAb

MacromoleculeName: Chimeric Nav1.7-NavAb / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Aliarcobacter butzleri RM4018 (bacteria)
Molecular weightTheoretical: 34.618094 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDYKDDDDKG SLVPRGSHMY LRITNIVESS FFTKFIIYLI VLNMVTMMVE KEGQSQHMTE VLYWINVVFI ILFTIEIILR IYVHRISFF KDPWSLFDFV VVIISIVGMF LADLIETYFV SPTLFRVIRL ARIGRILRLV TAVPQMRKIV SALISVIPGM L SVIALMTL ...String:
MDYKDDDDKG SLVPRGSHMY LRITNIVESS FFTKFIIYLI VLNMVTMMVE KEGQSQHMTE VLYWINVVFI ILFTIEIILR IYVHRISFF KDPWSLFDFV VVIISIVGMF LADLIETYFV SPTLFRVIRL ARIGRILRLV TAVPQMRKIV SALISVIPGM L SVIALMTL FFYIFAIMAT QLFGERFPEW FGTLGESFYT LFQVMTLESW SMGIVRPLME VYPYAWVFFI PFIFVVTFVM IN LVVAIIV DAMAILNQKE EQHIIDEVQS HEDNINNEII KLREEIVELK ELIKTSLKN

-
Macromolecule #2: Fab 7A9 heavy chain

MacromoleculeName: Fab 7A9 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.523518 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG LVKPGGSLKL SCAASGFTFS NYAMSWVRQT PEKRLEWVAT ISNGGRYTYY PDSVKGRFTI SRDNAKNSLY LQMSSLRSE DTAMYYCARH LYRYDVGGAL DYWGQGTSVT VSSAKTTAPS VYPLAPVCGD TTGSSVTLGC LVKGYFPEPV T LTWNSGSL ...String:
EVQLVESGGG LVKPGGSLKL SCAASGFTFS NYAMSWVRQT PEKRLEWVAT ISNGGRYTYY PDSVKGRFTI SRDNAKNSLY LQMSSLRSE DTAMYYCARH LYRYDVGGAL DYWGQGTSVT VSSAKTTAPS VYPLAPVCGD TTGSSVTLGC LVKGYFPEPV T LTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVTSSTWP SQSITCNVAH PASSTKVDKK IEPRGPTIKP

-
Macromolecule #3: Fab 7A9 light chain

MacromoleculeName: Fab 7A9 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.48391 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EIVLTQSPAL MAASPGEKVT ITCSVSLSIS SSNLFWYQQK SETSPKPWIY GTSKLASGVP VRFSGSGSGT SYSLTISSME AEDAATYYC QQWSSHSFTF GGGTKLEIKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD ...String:
EIVLTQSPAL MAASPGEKVT ITCSVSLSIS SSNLFWYQQK SETSPKPWIY GTSKLASGVP VRFSGSGSGT SYSLTISSME AEDAATYYC QQWSSHSFTF GGGTKLEIKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD SKDSTYSMSS TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNEC

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
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

-
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

-
Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 391335
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC

-
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
Output model

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

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more