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- PDB-8e15: A computationally stabilized hMPV F protein -

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Basic information

Entry
Database: PDB / ID: 8.0E+15
TitleA computationally stabilized hMPV F protein
Components
  • F1 protein with Fibritin peptide
  • F2 protein
KeywordsVIRAL PROTEIN / Human metapneumovirus / fusion protein / computational stabilization
Function / homology
Function and homology information


fusion of virus membrane with host plasma membrane / host cell plasma membrane / virion membrane / plasma membrane
Similarity search - Function
Precursor fusion glycoprotein F0, Paramyxoviridae / Fusion glycoprotein F0 / Fibritin C-terminal / Fibritin C-terminal region
Similarity search - Domain/homology
Fibritin / Fusion glycoprotein F0
Similarity search - Component
Biological speciesHuman metapneumovirus
Enterobacteria phage T2 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.41 Å
AuthorsHuang, J. / Gonzalez, K. / Mousa, J. / Strauch, E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI140245 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R01AI143865 United States
CitationJournal: bioRxiv / Year: 2023
Title: A general computational design strategy for stabilizing viral class I fusion proteins.
Authors: Karen J Gonzalez / Jiachen Huang / Miria F Criado / Avik Banerjee / Stephen Tompkins / Jarrod J Mousa / Eva-Maria Strauch /
Abstract: Many pathogenic viruses, including influenza virus, Ebola virus, coronaviruses, and Pneumoviruses, rely on class I fusion proteins to fuse viral and cellular membranes. To drive the fusion process, ...Many pathogenic viruses, including influenza virus, Ebola virus, coronaviruses, and Pneumoviruses, rely on class I fusion proteins to fuse viral and cellular membranes. To drive the fusion process, class I fusion proteins undergo an irreversible conformational change from a metastable prefusion state to an energetically more favorable and stable postfusion state. An increasing amount of evidence exists highlighting that antibodies targeting the prefusion conformation are the most potent. However, many mutations have to be evaluated before identifying prefusion-stabilizing substitutions. We therefore established a computational design protocol that stabilizes the prefusion state while destabilizing the postfusion conformation. As a proof of concept, we applied this principle to the fusion protein of the RSV, hMPV, and SARS-CoV-2 viruses. For each protein, we tested less than a handful of designs to identify stable versions. Solved structures of designed proteins from the three different viruses evidenced the atomic accuracy of our approach. Furthermore, the immunological response of the RSV F design compared to a current clinical candidate in a mouse model. While the parallel design of two conformations allows identifying and selectively modifying energetically less optimized positions for one conformation, our protocol also reveals diverse molecular strategies for stabilization. We recaptured many approaches previously introduced manually for the stabilization of viral surface proteins, such as cavity-filling, optimization of polar interactions, as well as postfusion-disruptive strategies. Using our approach, it is possible to focus on the most impacting mutations and potentially preserve the immunogen as closely as possible to its native version. The latter is important as sequence re-design can cause perturbations to B and T cell epitopes. Given the clinical significance of viruses using class I fusion proteins, our algorithm can substantially contribute to vaccine development by reducing the time and resources needed to optimize these immunogens.
History
DepositionAug 9, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
F: F2 protein
G: F1 protein with Fibritin peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,7155
Polymers57,6862
Non-polymers1,0293
Water19811
1
F: F2 protein
G: F1 protein with Fibritin peptide
hetero molecules

F: F2 protein
G: F1 protein with Fibritin peptide
hetero molecules

F: F2 protein
G: F1 protein with Fibritin peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)176,14615
Polymers173,0596
Non-polymers3,0879
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation5_555z,x,y1
crystal symmetry operation9_555y,z,x1
Buried area43220 Å2
ΔGint-189 kcal/mol
Surface area49790 Å2
MethodPISA
Unit cell
Length a, b, c (Å)178.191, 178.191, 178.191
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number199
Space group name H-MI213
Space group name HallI2b2c3
Symmetry operation#1: x,y,z
#2: z,x,y
#3: y,z,x
#4: -y,-z+1/2,x
#5: z,-x,-y+1/2
#6: -y+1/2,z,-x
#7: -z,-x+1/2,y
#8: -z+1/2,x,-y
#9: y,-z,-x+1/2
#10: x,-y,-z+1/2
#11: -x+1/2,y,-z
#12: -x,-y+1/2,z
#13: x+1/2,y+1/2,z+1/2
#14: z+1/2,x+1/2,y+1/2
#15: y+1/2,z+1/2,x+1/2
#16: -y+1/2,-z+1,x+1/2
#17: z+1/2,-x+1/2,-y+1
#18: -y+1,z+1/2,-x+1/2
#19: -z+1/2,-x+1,y+1/2
#20: -z+1,x+1/2,-y+1/2
#21: y+1/2,-z+1/2,-x+1
#22: x+1/2,-y+1/2,-z+1
#23: -x+1,y+1/2,-z+1/2
#24: -x+1/2,-y+1,z+1/2

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Components

#1: Protein F2 protein


Mass: 11687.245 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human metapneumovirus / Cell line (production host): 293 / Production host: Homo sapiens (human) / References: UniProt: Q8B9P0
#2: Protein F1 protein with Fibritin peptide


Mass: 45999.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human metapneumovirus, (gene. exp.) Enterobacteria phage T2 (virus)
Gene: F, wac, EcT2_00172 / Cell line (production host): 293 / Production host: Homo sapiens (human) / References: UniProt: Q8B9P0, UniProt: Q76VI8
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 11 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 4.09 Å3/Da / Density % sol: 69.91 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop
Details: 0.1 M Sodium acetate trihydrate pH 4.6, 2.0 M Sodium formate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Apr 21, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.41→47.62 Å / Num. obs: 36363 / % possible obs: 99.91 % / Redundancy: 2 % / Biso Wilson estimate: 66.92 Å2 / CC1/2: 0.999 / CC star: 1 / Net I/σ(I): 9.18
Reflection shellResolution: 2.41→2.496 Å / Redundancy: 2 % / Mean I/σ(I) obs: 0.78 / Num. unique obs: 3606 / CC1/2: 0.593 / CC star: 0.863 / % possible all: 99.86

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5wb0

5wb0


Resolution: 2.41→47.62 Å / SU ML: 0.3364 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.647
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2487 1871 5.15 %
Rwork0.2036 34469 -
obs0.2058 36340 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 70.9 Å2
Refinement stepCycle: LAST / Resolution: 2.41→47.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3360 0 67 11 3438
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00923475
X-RAY DIFFRACTIONf_angle_d1.01724714
X-RAY DIFFRACTIONf_chiral_restr0.0567573
X-RAY DIFFRACTIONf_plane_restr0.008604
X-RAY DIFFRACTIONf_dihedral_angle_d16.05451279
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.41-2.470.35131400.31092649X-RAY DIFFRACTION99.82
2.48-2.550.30051500.28492590X-RAY DIFFRACTION99.96
2.55-2.630.30371470.29982632X-RAY DIFFRACTION99.89
2.63-2.720.35691490.30322615X-RAY DIFFRACTION99.93
2.73-2.830.34191720.30792614X-RAY DIFFRACTION99.96
2.83-2.960.31121300.27312644X-RAY DIFFRACTION99.86
2.96-3.120.30281620.25732627X-RAY DIFFRACTION99.82
3.12-3.310.29011360.25482645X-RAY DIFFRACTION99.93
3.31-3.570.27891350.22372647X-RAY DIFFRACTION100
3.57-3.930.25841450.20012654X-RAY DIFFRACTION99.96
3.93-4.490.24041230.17012689X-RAY DIFFRACTION100
4.5-5.660.18571530.16592683X-RAY DIFFRACTION100
5.67-47.620.19771290.16422780X-RAY DIFFRACTION99.97

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