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- PDB-9c1u: Cryo-EM Structure of a Tm1C Fibril -

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Basic information

Entry
Database: PDB / ID: 9c1u
TitleCryo-EM Structure of a Tm1C Fibril
ComponentsTropomyosin 1 I/C
KeywordsPROTEIN FIBRIL
Function / homologyTropomyosin / Tropomyosin / GH09289p
Function and homology information
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.31 Å
AuthorsFonda, B.D. / Kato, M. / Li, Y. / Murray, D.T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35142892 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP220582 United States
CitationJournal: Protein Sci / Year: 2024
Title: Cryo-EM and solid state NMR together provide a more comprehensive structural investigation of protein fibrils.
Authors: Blake D Fonda / Masato Kato / Yang Li / Dylan T Murray /
Abstract: The tropomyosin 1 isoform I/C C-terminal domain (Tm1-LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo-EM) and solid state nuclear magnetic resonance (NMR). This study ...The tropomyosin 1 isoform I/C C-terminal domain (Tm1-LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo-EM) and solid state nuclear magnetic resonance (NMR). This study demonstrates the complementary nature of these two structural biology techniques. Chemical shift assignments from solid state NMR are used to determine the secondary structure at the level of individual amino acids, which is faithfully seen in cryo-EM reconstructions. Additionally, solid state NMR demonstrates that the region not observed in the reconstructed cryo-EM density is primarily in a highly mobile random coil conformation rather than adopting multiple rigid conformations. Overall, this study illustrates the benefit of investigations combining cryo-EM and solid state NMR to investigate protein fibril structure.
History
DepositionMay 29, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tropomyosin 1 I/C
B: Tropomyosin 1 I/C
C: Tropomyosin 1 I/C
D: Tropomyosin 1 I/C
E: Tropomyosin 1 I/C
F: Tropomyosin 1 I/C
G: Tropomyosin 1 I/C
H: Tropomyosin 1 I/C
I: Tropomyosin 1 I/C
J: Tropomyosin 1 I/C
K: Tropomyosin 1 I/C
L: Tropomyosin 1 I/C


Theoretical massNumber of molelcules
Total (without water)89,04612
Polymers89,04612
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Tropomyosin 1 I/C / LD11194p / Tropomyosin 1 / isoform C / isoform I / GH09289p


Mass: 7420.535 Da / Num. of mol.: 12 / Fragment: C-terminal domain, residues 373-441
Source method: isolated from a genetically manipulated source
Details: SY-tagged Tm1 I/C alternative isoform / Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Tm1, 1305/10, 2299, BcDNA:GH09289, BcDNA:LD37158, BcDNA:SD21996, chr3R:11122272..11122408, cTM, cTm, cTmII, Dm Tm1, Dm TmH33, Dm TmH34, Dmel\CG4898, DmTm1, l(3)02299, l(3)S130510, l(3)s2958, ...Gene: Tm1, 1305/10, 2299, BcDNA:GH09289, BcDNA:LD37158, BcDNA:SD21996, chr3R:11122272..11122408, cTM, cTm, cTmII, Dm Tm1, Dm TmH33, Dm TmH34, Dmel\CG4898, DmTm1, l(3)02299, l(3)S130510, l(3)s2958, mTmII, PmI, region 3, TM, Tm, TM1, tm1, TmH, TmH-33, TmH-34, TmH33, TmH34, TMII, TmII, tmII, Tmr33, Tmr34, TnH, TnH-33, TnH-34, tropomyosin, CG4898, Dmel_CG4898,
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q95TA3
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Tropomyosin 1 I/C C-terminal Domain / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 7.41557 kDa/nm / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMTris-HCl1
2200 mMsodium chloride1
30.5 mMethylenediaminetetraacetic acid1
40.1 mMphenylmethylsulfonyl fluoride1
520 mMB-mercaptoethanol1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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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 defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 2.58 ° / Axial rise/subunit: 4.69 Å / Axial symmetry: C1
3D reconstructionResolution: 2.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17145 / Num. of class averages: 1 / Symmetry type: HELICAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0032376
ELECTRON MICROSCOPYf_angle_d0.4573180
ELECTRON MICROSCOPYf_dihedral_angle_d5.805312
ELECTRON MICROSCOPYf_chiral_restr0.046360
ELECTRON MICROSCOPYf_plane_restr0.001432

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