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- EMDB-1995: Electron density map of a composite coiled-coil fibril comprising... -

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Entry
Database: EMDB / ID: EMD-1995
TitleElectron density map of a composite coiled-coil fibril comprising multiple self-assembling fibre peptides.
Map dataThis is a map of a coiled coil derived from a micrograph of a 3D protein fibre processed using 2dx
Sample
  • Sample: Self-assembling peptide fibre
  • Protein or peptide: KIAALKQKIASLKQEIDALEYENDALEQ
  • Protein or peptide: KIRRLKQKNARLKQEIAALEYEIAALEQ
KeywordsCryoTEM / coiled coil / fibrous proteins / protein design / self-assembly
Biological speciessynthetic construct (others)
Methodelectron crystallography / cryo EM / Resolution: 8.0 Å
AuthorsSharp TH / Bruning M / Mantell J / Sessions RB / Thomson AR / Zaccai NR / Brady RL / Verkade P / Woolfson DN
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Cryo-transmission electron microscopy structure of a gigadalton peptide fiber of de novo design.
Authors: Thomas H Sharp / Marc Bruning / Judith Mantell / Richard B Sessions / Andrew R Thomson / Nathan R Zaccai / R Leo Brady / Paul Verkade / Derek N Woolfson /
Abstract: Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address ...Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address difficulties in studying and understanding natural systems, and to provide routes to new biomaterials with potential applications in nanotechnology and medicine. We have designed a self-assembling fiber system, the SAFs, in which two small α-helical peptides are programmed to form a dimeric coiled coil and assemble in a controlled manner. The resulting fibers are tens of nm wide and tens of μm long, and, therefore, comprise millions of peptides to give gigadalton supramolecular structures. Here, we describe the structure of the SAFs determined to approximately 8 Å resolution using cryotransmission electron microscopy. Individual micrographs show clear ultrastructure that allowed direct interpretation of the packing of individual α-helices within the fibers, and the construction of a 3D electron density map. Furthermore, a model was derived using the cryotransmission electron microscopy data and side chains taken from a 2.3 Å X-ray crystal structure of a peptide building block incapable of forming fibers. This was validated using single-particle analysis techniques, and was stable in prolonged molecular-dynamics simulation, confirming its structural viability. The level of self-assembly and self-organization in the SAFs is unprecedented for a designed peptide-based material, particularly for a system of considerably reduced complexity compared with natural proteins. This structural insight is a unique high-resolution description of how α-helical fibrils pack into larger protein fibers, and provides a basis for the design and engineering of future biomaterials.
History
DepositionNov 19, 2011-
Header (metadata) releaseDec 16, 2011-
Map releaseAug 15, 2012-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 15.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 15.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

SAF.png

Downloads & links

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Map

FileDownload / File: emd_1995.map.gz / Format: CCP4 / Size: 4.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a map of a coiled coil derived from a micrograph of a 3D protein fibre processed using 2dx
Voxel sizeX=Y=Z: 0.627 Å
Density
Contour LevelBy AUTHOR: 15.5 / Movie #1: 15.5
Minimum - Maximum-65.063873290000004 - 32.860794069999997
Average (Standard dev.)-13.47298717 (±25.133878710000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions101101126
Spacing101101126
CellA: 63.326996 Å / B: 63.326996 Å / C: 79.002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6270.6270.627
M x/y/z101101126
origin x/y/z0.0000.0000.000
length x/y/z63.32763.32779.002
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS101101126
D min/max/mean-65.06432.861-13.473

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Supplemental data

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Sample components

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Entire : Self-assembling peptide fibre

EntireName: Self-assembling peptide fibre
Components
  • Sample: Self-assembling peptide fibre
  • Protein or peptide: KIAALKQKIASLKQEIDALEYENDALEQ
  • Protein or peptide: KIRRLKQKNARLKQEIAALEYEIAALEQ

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Supramolecule #1000: Self-assembling peptide fibre

SupramoleculeName: Self-assembling peptide fibre / type: sample / ID: 1000 / Details: Samples contains approximately 30,000,000 peptides
Oligomeric state: Many millions of peptides self-assemble to form a gigadalton peptide fibre
Number unique components: 2

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Macromolecule #1: KIAALKQKIASLKQEIDALEYENDALEQ

MacromoleculeName: KIAALKQKIASLKQEIDALEYENDALEQ / type: protein_or_peptide / ID: 1 / Name.synonym: Self-assembling fibre
Details: Peptides were synthesized by microwave-assisted solid-phase peptide synthesis using standard HBTU activation. They heterodimerize to form offset dimeric coiled coils with complementary ...Details: Peptides were synthesized by microwave-assisted solid-phase peptide synthesis using standard HBTU activation. They heterodimerize to form offset dimeric coiled coils with complementary sticky ends that assemble to form extended coiled coil fibrils. These fibrils pack laterally to generate large proteinaceous fibres on average 82 nm in diameter and 42 micrometers in length.
Recombinant expression: No / Database: NCBI
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.17 KDa

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Macromolecule #2: KIRRLKQKNARLKQEIAALEYEIAALEQ

MacromoleculeName: KIRRLKQKNARLKQEIAALEYEIAALEQ / type: protein_or_peptide / ID: 2 / Name.synonym: Self-assembling fibre
Details: Peptides were synthesized by microwave-assisted solid-phase peptide synthesis using standard HBTU activation. They heterodimerize to form offset dimeric coiled coils with complementary ...Details: Peptides were synthesized by microwave-assisted solid-phase peptide synthesis using standard HBTU activation. They heterodimerize to form offset dimeric coiled coils with complementary sticky ends that assemble to form extended coiled coil fibrils. These fibrils pack laterally to generate large proteinaceous fibres on average 82 nm in diameter and 42 micrometers in length.
Recombinant expression: No / Database: NCBI
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.32 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration0.325 mg/mL
BufferpH: 7.4 / Details: 10 mM MOPS
GridDetails: Lacey-carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 98 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot 1 sec.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 0 °
DetailsLow dose software used
DateApr 20, 2010
Image recordingDigitization - Sampling interval: 11 µm / Number real images: 1 / Average electron dose: 10 e/Å2
Tilt angle min0
Tilt angle max0

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Image processing

Crystal parametersUnit cell - A: 20.8 Å / Unit cell - B: 20.8 Å / Unit cell - C: 125.4 Å / Unit cell - γ: 120 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Plane group: P 1
CTF correctionDetails: Each image
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: OTHER / Software - Name: 2dx, BHP

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