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- PDB-3ra3: Crystal structure of a section of a de novo design gigaDalton pro... -

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Basic information

Entry
Database: PDB / ID: 3ra3
TitleCrystal structure of a section of a de novo design gigaDalton protein fibre
Components
  • p1c
  • p2f
KeywordsDE NOVO PROTEIN / coiled coil domain / fiber / KIH interactions / synthetic biology / helical reconstruction
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.31 Å
AuthorsZaccai, N.R. / Sharp, T.H. / Bruning, M. / Woolfson, D.N. / Brady, R.L.
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
DepositionMar 27, 2011Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Aug 8, 2012Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2013Group: Database references
Revision 1.2Mar 26, 2025Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: p1c
B: p2f
D: p2f
E: p1c
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,2625
Polymers13,2394
Non-polymers231
Water61334
1
A: p1c
B: p2f


Theoretical massNumber of molelcules
Total (without water)6,6192
Polymers6,6192
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1390 Å2
ΔGint-12 kcal/mol
Surface area4400 Å2
MethodPISA
2
D: p2f
E: p1c
hetero molecules


Theoretical massNumber of molelcules
Total (without water)6,6423
Polymers6,6192
Non-polymers231
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1400 Å2
ΔGint-13 kcal/mol
Surface area4500 Å2
MethodPISA
Unit cell
Length a, b, c (Å)45.082, 45.082, 67.593
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number145
Space group name H-MP32

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Components

#1: Protein/peptide p1c


Mass: 3287.498 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Peptide synthesis was carried out according to standard Fmoc SPPS protocols
#2: Protein/peptide p2f


Mass: 3331.928 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Peptide synthesis was carried out according to standard Fmoc SPPS protocols
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 34 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3 Å3/Da / Density % sol: 58.94 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 4.5
Details: 0.2M zinc acetate, 0.1M sodium acetate, 10%(w/v) PEG 3K, pH 4.5, VAPOR DIFFUSION, SITTING DROP, temperature 291K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 1.7 Å
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.7 Å / Relative weight: 1
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H, K, L10.819
11h+k,-k,-l20.181
ReflectionResolution: 2.3→25 Å / Num. obs: 6777 / % possible obs: 99.8 % / Redundancy: 8.2 % / Rmerge(I) obs: 0.102 / Net I/σ(I): 21
Reflection shellResolution: 2.3→2.38 Å / Redundancy: 5 % / Rmerge(I) obs: 0.606 / Mean I/σ(I) obs: 1.95 / Num. unique all: 1388 / % possible all: 98.8

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Phasing

PhasingMethod: SAD

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Processing

Software
NameVersionClassificationNB
SHELXphasing
REFMAC5.5.0109refinement
PDB_EXTRACT3.1data extraction
DENZOdata reduction
SCALEPACKdata scaling
SHELXDphasing
RefinementMethod to determine structure: SAD / Resolution: 2.31→22.54 Å / Cor.coef. Fo:Fc: 0.945 / Cor.coef. Fo:Fc free: 0.902 / Occupancy max: 1 / Occupancy min: 1 / SU B: 7.058 / SU ML: 0.166 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.047 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: U VALUES: REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.248 701 10.4 %RANDOM
Rwork0.1882 ---
obs0.1941 6762 99.81 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso max: 85.03 Å2 / Biso mean: 45.7517 Å2 / Biso min: 26.03 Å2
Baniso -1Baniso -2Baniso -3
1-5.04 Å20 Å20 Å2
2--5.04 Å20 Å2
3----10.08 Å2
Refinement stepCycle: LAST / Resolution: 2.31→22.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms857 0 1 34 892
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.022857
X-RAY DIFFRACTIONr_angle_refined_deg1.8162.031140
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.5265102
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.38826.66742
X-RAY DIFFRACTIONr_dihedral_angle_3_deg21.48715188
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.957156
X-RAY DIFFRACTIONr_chiral_restr0.1040.2133
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.02606
X-RAY DIFFRACTIONr_mcbond_it1.031.5526
X-RAY DIFFRACTIONr_mcangle_it2.0342827
X-RAY DIFFRACTIONr_scbond_it3.5613331
X-RAY DIFFRACTIONr_scangle_it6.2624.5313
LS refinement shellResolution: 2.307→2.367 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.364 49 -
Rwork0.231 464 -
all-513 -
obs--97.53 %

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