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Basic information

Entry
Database: PDB / ID: 6ibg
TitleBacteriophage G20c portal protein crystal structure for construct with intact N-terminus
ComponentsPortal protein
KeywordsVIRAL PROTEIN / bacteriophage / thermophage / caudovirales / siphoviridae / capsid / auxiliary / HK97 / virus
Function / homology: / Phage P23-45 portal protein / viral capsid / Portal protein
Function and homology information
Biological speciesThermus virus P23-45
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 1.95 Å
AuthorsBayfield, O.W. / Klimuk, E. / Winkler, D.C. / Hesketh, E.L. / Chechik, M. / Cheng, N. / Dykeman, E.C. / Minakhin, L. / Ranson, N.A. / Severinov, K. ...Bayfield, O.W. / Klimuk, E. / Winkler, D.C. / Hesketh, E.L. / Chechik, M. / Cheng, N. / Dykeman, E.C. / Minakhin, L. / Ranson, N.A. / Severinov, K. / Steven, A.C. / Antson, A.A.
Funding support United Kingdom, United States, Russian Federation, 6items
OrganizationGrant numberCountry
Wellcome Trust206377 United Kingdom
Wellcome Trust103460 United Kingdom
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS) United States
Russian Foundation for Basic Research16-34-60137 Russian Federation
Biotechnology and Biological Sciences Research CouncilBB/L021250/1 United Kingdom
Wellcome Trust108466 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids.
Authors: Oliver W Bayfield / Evgeny Klimuk / Dennis C Winkler / Emma L Hesketh / Maria Chechik / Naiqian Cheng / Eric C Dykeman / Leonid Minakhin / Neil A Ranson / Konstantin Severinov / Alasdair C ...Authors: Oliver W Bayfield / Evgeny Klimuk / Dennis C Winkler / Emma L Hesketh / Maria Chechik / Naiqian Cheng / Eric C Dykeman / Leonid Minakhin / Neil A Ranson / Konstantin Severinov / Alasdair C Steven / Alfred A Antson /
Abstract: Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic ...Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.
History
DepositionNov 30, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 27, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
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Revision 1.2Mar 6, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 30, 2019Group: Data collection / Database references ...Data collection / Database references / Source and taxonomy / Structure summary
Category: entity_name_com / entity_src_gen ...entity_name_com / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _entity_src_gen.pdbx_gene_src_gene / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id ..._entity_src_gen.pdbx_gene_src_gene / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.pdbx_db_accession
Revision 1.4Mar 30, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 1.5Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Portal protein
B: Portal protein
C: Portal protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,5206
Polymers148,1663
Non-polymers3553
Water11,007611
1
A: Portal protein
B: Portal protein
C: Portal protein
hetero molecules

A: Portal protein
B: Portal protein
C: Portal protein
hetero molecules

A: Portal protein
B: Portal protein
C: Portal protein
hetero molecules

A: Portal protein
B: Portal protein
C: Portal protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)594,08224
Polymers592,66412
Non-polymers1,41812
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation3_555-y,x,z1
crystal symmetry operation4_555y,-x,z1
Buried area135590 Å2
ΔGint-942 kcal/mol
Surface area170620 Å2
MethodPISA
Unit cell
Length a, b, c (Å)159.081, 159.081, 116.913
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number79
Space group name H-MI4
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13B
23C

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: LEU / Beg label comp-ID: LEU / Refine code: _

Dom-IDEns-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ALAALAAA26 - 43726 - 437
21ALAALABB26 - 43726 - 437
12ARGARGAA26 - 43426 - 434
22ARGARGCC26 - 43426 - 434
13ARGARGBB26 - 43426 - 434
23ARGARGCC26 - 43426 - 434

NCS ensembles :
ID
1
2
3

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Components

#1: Protein Portal protein / Gene product 86 / gp86


Mass: 49388.648 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus virus P23-45 / Gene: P23p86 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A7XXB9
#2: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL


Mass: 118.174 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 611 / Source method: isolated from a natural source / Formula: H2O

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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: 2.5 Å3/Da / Density % sol: 50.72 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 13.3mg/ml protein, 2mg/ml 42-amino acid peptide kdgykfelaeerpsklkheesvmslveddftdlelanrafsa, 20 mM Tris-HCl pH 7.5, 1 M NaCl, 5 % v/v glycerol. Reservoir: 40 % MPD v/v, 200 mM NaH2PO4

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Data collection

DiffractionMean temperature: 120 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.92819 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jun 19, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.92819 Å / Relative weight: 1
ReflectionResolution: 1.95→94.2 Å / Num. obs: 105514 / % possible obs: 99.8 % / Redundancy: 6.8 % / CC1/2: 0.999 / Rmerge(I) obs: 0.103 / Net I/av σ(I): 6.2 / Net I/σ(I): 6.2
Reflection shellResolution: 1.95→1.98 Å / Redundancy: 7 % / Rmerge(I) obs: 1.199 / Mean I/σ(I) obs: 0.6 / Num. unique obs: 7752 / CC1/2: 0.626 / % possible all: 99.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
DIALSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementResolution: 1.95→10 Å / Cor.coef. Fo:Fc: 0.96 / Cor.coef. Fo:Fc free: 0.949 / SU B: 10.818 / SU ML: 0.126 / Cross valid method: THROUGHOUT / ESU R: 0.298 / ESU R Free: 0.133 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.21613 1013 1 %RANDOM
Rwork0.15822 ---
obs0.15879 104315 99.58 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 34.453 Å2
Baniso -1Baniso -2Baniso -3
1-0.11 Å20 Å20 Å2
2--0.11 Å20 Å2
3----0.22 Å2
Refinement stepCycle: 1 / Resolution: 1.95→10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9621 0 24 611 10256
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0139963
X-RAY DIFFRACTIONr_bond_other_d0.0010.0179555
X-RAY DIFFRACTIONr_angle_refined_deg1.3671.63413559
X-RAY DIFFRACTIONr_angle_other_deg1.2161.57322144
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.24851265
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.50222.769484
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.859151707
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.1691554
X-RAY DIFFRACTIONr_chiral_restr0.0610.21318
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0211095
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022028
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.8512.4344967
X-RAY DIFFRACTIONr_mcbond_other2.8512.4344966
X-RAY DIFFRACTIONr_mcangle_it3.784.16212
X-RAY DIFFRACTIONr_mcangle_other3.784.16213
X-RAY DIFFRACTIONr_scbond_it3.6832.8984996
X-RAY DIFFRACTIONr_scbond_other3.6822.8984997
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.6164.7027331
X-RAY DIFFRACTIONr_long_range_B_refined5.35624.72310958
X-RAY DIFFRACTIONr_long_range_B_other5.28124.49110869
X-RAY DIFFRACTIONr_rigid_bond_restr2.398319518
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.07 Å / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumber
11A12252
12B12252
21A12143
22C12143
31B12193
32C12193
LS refinement shellResolution: 1.95→2.001 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.419 52 -
Rwork0.354 7662 -
obs--99.42 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.5636-0.09220.13813.0325-0.58370.48040.0381-0.27090.05240.641-0.0732-0.0709-0.07480.02720.03510.281-0.0093-0.03850.2648-0.02130.048124.63322.380531.5133
25.6274-0.7431-0.27963.78490.10277.4386-0.01580.11530.6464-0.1558-0.0168-0.1472-0.8466-0.06980.03260.1529-0.0122-0.03750.10610.03260.09795.350230.8947-51.4008
35.7047-0.5502-0.96475.2263-1.12755.65460.0022-0.00670.36630.0939-0.018-0.2623-0.30090.16960.01580.0398-0.0467-0.02460.06620.03410.072219.759723.8658-51.5285
44.8657-0.71831.55586.32740.22656.43740.00410.10730.1758-0.16410.0288-0.636-0.11360.6321-0.03290.0958-0.02060.01840.11630.02510.091229.341110.7704-51.4998
51.36290.41610.1591.31370.21220.6851-0.0375-0.04730.13470.05780.0305-0.2345-0.1580.20640.0070.0514-0.0471-0.03180.0693-0.00680.08334.52227.75261.3627
61.69730.17080.31631.1240.09110.7722-0.0017-0.02860.0260.0652-0.0005-0.3388-0.01850.28570.00220.0146-0.0114-0.02870.12040.00740.112443.83556.77821.3433
71.4429-0.2290.24181.1661-0.08320.73640.0135-0.0513-0.09110.0473-0.0178-0.29050.10450.23840.00440.02450.0302-0.02010.09360.01840.095741.3396-16.01531.4009
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A382 - 437
2X-RAY DIFFRACTION1B382 - 436
3X-RAY DIFFRACTION1C382 - 435
4X-RAY DIFFRACTION2A243 - 288
5X-RAY DIFFRACTION3B243 - 288
6X-RAY DIFFRACTION4C243 - 288
7X-RAY DIFFRACTION5A26 - 242
8X-RAY DIFFRACTION5A289 - 381
9X-RAY DIFFRACTION6B26 - 242
10X-RAY DIFFRACTION6B289 - 381
11X-RAY DIFFRACTION7C26 - 242
12X-RAY DIFFRACTION7C289 - 381

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