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- EMDB-9012: Sub-2 Angstrom Ewald Curvature Corrected Single-Particle Cryo-EM ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9012
TitleSub-2 Angstrom Ewald Curvature Corrected Single-Particle Cryo-EM Reconstruction of AAV-2 L336C
Map dataFinal map reconstructed with a box of 800, clipped to 360, and then resampled to 720 pixels
Sample
  • Virus: Adeno-associated virus - 2
    • Protein or peptide: Capsid protein VP1
  • Ligand: water
KeywordsAAV-2 L336C / Gene therapy / Ewald sphere curvature correction / Per particle CTF / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell nucleolus / T=1 icosahedral viral capsid / clathrin-dependent endocytosis of virus by host cell / structural molecule activity / virion attachment to host cell
Similarity search - Function
Phospholipase A2-like domain / Phospholipase A2-like domain / Parvovirus coat protein VP2 / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP2 / Capsid/spike protein, ssDNA virus
Similarity search - Domain/homology
Biological speciesAdeno-associated virus 2 Srivastava/1982 / Adeno-associated virus - 2
Methodsingle particle reconstruction / cryo EM / Resolution: 1.86 Å
AuthorsTan YZ / Aiyer S
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI136680-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM109524 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM033050 United States
CitationJournal: Nat Commun / Year: 2018
Title: Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant.
Authors: Yong Zi Tan / Sriram Aiyer / Mario Mietzsch / Joshua A Hull / Robert McKenna / Joshua Grieger / R Jude Samulski / Timothy S Baker / Mavis Agbandje-McKenna / Dmitry Lyumkis /
Abstract: Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures ...Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we exploit several technical advances to improve resolution, including per-particle contrast transfer function (CTF) refinement and correction for Ewald sphere curvature. The latter is demonstrated with several experimental samples and should become more standard as resolutions increase or at lower microscope accelerating voltages. The combined application of the described methods to micrographs recorded on a Titan Krios enables structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle. The resulting structural details provide an improved model for understanding the biology of AAV that will guide future vector development for gene therapy.
History
DepositionJul 31, 2018-
Header (metadata) releaseAug 15, 2018-
Map releaseAug 15, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6e9d
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6e9d
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9012.png

Downloads & links

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Map

FileDownload / File: emd_9012.map.gz / Format: CCP4 / Size: 1.4 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map reconstructed with a box of 800, clipped to 360, and then resampled to 720 pixels
Voxel sizeX=Y=Z: 0.394 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.22251815 - 0.41616842
Average (Standard dev.)0.0019748327 (±0.02993493)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions720720720
Spacing720720720
CellA=B=C: 283.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.3940.3940.394
M x/y/z720720720
origin x/y/z0.0000.0000.000
length x/y/z283.680283.680283.680
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS720720720
D min/max/mean-0.2230.4160.002

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Supplemental data

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Additional map: Raw 3D FSC volume generated from the half maps

Fileemd_9012_additional.map
AnnotationRaw 3D FSC volume generated from the half maps
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Second half map reconstructed with a box of...

Fileemd_9012_half_map_1.map
AnnotationSecond half map reconstructed with a box of 800, clipped to 360 pixels
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: First half map reconstructed with a box of 800, clipped to 360 pixels

Fileemd_9012_half_map_2.map
AnnotationFirst half map reconstructed with a box of 800, clipped to 360 pixels
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Adeno-associated virus - 2

EntireName: Adeno-associated virus - 2
Components
  • Virus: Adeno-associated virus - 2
    • Protein or peptide: Capsid protein VP1
  • Ligand: water

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Supramolecule #1: Adeno-associated virus - 2

SupramoleculeName: Adeno-associated virus - 2 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1 / NCBI-ID: 10804 / Sci species name: Adeno-associated virus - 2 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.9 MDa
Virus shellShell ID: 1 / Name: Icosahedral capsid / Diameter: 250.0 Å / T number (triangulation number): 1

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Macromolecule #1: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus 2 Srivastava/1982
Molecular weightTheoretical: 82.021336 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNP YLKYNHADAE FQERLKEDTS FGGNLGRAVF QAKKRVLEPL GLVEEPVKTA PGKKRPVEHS PVEPDSSSGT G KAGQQPAR ...String:
MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNP YLKYNHADAE FQERLKEDTS FGGNLGRAVF QAKKRVLEPL GLVEEPVKTA PGKKRPVEHS PVEPDSSSGT G KAGQQPAR KRLNFGQTGD ADSVPDPQPL GQPPAAPSGL GTNTMATGSG APMADNNEGA DGVGNSSGNW HCDSTWMGDR VI TTSTRTW ALPTYNNHLY KQISSQSGAS NDNHYFGYST PWGYFDFNRF HCHFSPRDWQ RLINNNWGFR PKRLNFKLFN IQV KEVTQN DGTTTIANNC TSTVQVFTDS EYQLPYVLGS AHQGCLPPFP ADVFMVPQYG YLTLNNGSQA VGRSSFYCLE YFPS QMLRT GNNFTFSYTF EDVPFHSSYA HSQSLDRLMN PLIDQYLYYL SRTNTPSGTT TQSRLQFSQA GASDIRDQSR NWLPG PCYR QQRVSKTSAD NNNSEYSWTG ATKYHLNGRD SLVNPGPAMA SHKDDEEKFF PQSGVLIFGK QGSEKTNVDI EKVMIT DEE EIRTTNPVAT EQYGSVSTNL QRGNRQAATA DVNTQGVLPG MVWQDRDVYL QGPIWAKIPH TDGHFHPSPL MGGFGLK HP PPQILIKNTP VPANPSTTFS AAKFASFITQ YSTGQVSVEI EWELQKENSK RWNPEIQYTS NYNKSVNVDF TVDTNGVY S EPRPIGTRYL TRNL

UniProtKB: Capsid protein VP1

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 11820 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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

Concentration1.7 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
2.0 mMMgCl2magnesium chloride
50.0 mMC8H18N2O4SHEPES
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 5 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: Double blotting was used to increase particle concentration. Sample was added to a plasma-cleaned (Gatan Solarus) grid and blotted away using Whatman grade 4 filter paper after 20 second wait time..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsBeam tilt in 8 directions +/- 5 mrad
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 5-19 / Number grids imaged: 1 / Number real images: 1317 / Average exposure time: 3.5 sec. / Average electron dose: 22.5 e/Å2 / Details: Stage shift mode for data collection
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 78194
Details: Template-based picking by generating template from a 2-D classification of an initial subset of data.
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generated using CryoSPARC.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 0.65)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Details: Frequency limited refinement / Number images used: 30515
Detailssuper-resolution mode

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Atomic model buiding 1

Initial modelPDB ID:

1lp3


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 61.9 / Target criteria: Correlation coefficient, EM ringer score
Output model

PDB-6e9d:
Sub-2 Angstrom Ewald Curvature-Corrected Single-Particle Cryo-EM Reconstruction of AAV-2 L336C

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