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- PDB-6anv: Crystal structure of anti-CRISPR protein AcrF1 -

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Basic information

Entry
Database: PDB / ID: 6anv
TitleCrystal structure of anti-CRISPR protein AcrF1
Componentsanti-CRISPR protein AcrF1 fused with C-terminal MBP tag
KeywordsIMMUNE SYSTEM / Type I-F CRISPR-Cas system: Csy Cascade: Structure: anti-CRISPR protein: Inhibition of Csy complex: Genome editing tool
Function / homology
Function and homology information


carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / outer membrane-bounded periplasmic space
Similarity search - Function
: / Anti-CRISPR protein Acr30-35/AcrF1 / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
alpha-maltotetraose / DI(HYDROXYETHYL)ETHER / TRIETHYLENE GLYCOL / Uncharacterized protein / Maltose/maltodextrin-binding periplasmic protein
Similarity search - Component
Biological speciesPseudomonas phage JBD30 (virus)
Escherichia coli O157:H7 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.265 Å
AuthorsYang, H. / Patel, D.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)GM104962 United States
Memorial Sloan-Kettering Cancer Center Core GrantP30CA008748 United States
CitationJournal: Cell / Year: 2017
Title: Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.
Authors: Tai Wei Guo / Alberto Bartesaghi / Hui Yang / Veronica Falconieri / Prashant Rao / Alan Merk / Edward T Eng / Ashleigh M Raczkowski / Tara Fox / Lesley A Earl / Dinshaw J Patel / Sriram Subramaniam /
Abstract: Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided ...Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.
History
DepositionAug 14, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 25, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / chem_comp / entity / entity_name_com / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_molecule_features / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _atom_site_anisotrop.U[1][1] / _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][2] / _atom_site_anisotrop.U[2][3] / _atom_site_anisotrop.U[3][3] / _atom_site_anisotrop.pdbx_auth_asym_id / _atom_site_anisotrop.pdbx_auth_atom_id / _atom_site_anisotrop.pdbx_auth_comp_id / _atom_site_anisotrop.pdbx_auth_seq_id / _atom_site_anisotrop.pdbx_label_asym_id / _atom_site_anisotrop.pdbx_label_atom_id / _atom_site_anisotrop.pdbx_label_comp_id / _atom_site_anisotrop.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _entity.pdbx_description / _entity.src_method / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_asym.entity_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Oct 4, 2023Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: anti-CRISPR protein AcrF1 fused with C-terminal MBP tag
B: anti-CRISPR protein AcrF1 fused with C-terminal MBP tag
hetero molecules


Theoretical massNumber of molelcules
Total (without water)102,89823
Polymers99,5462
Non-polymers3,35121
Water4,558253
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A: anti-CRISPR protein AcrF1 fused with C-terminal MBP tag
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,39611
Polymers49,7731
Non-polymers1,62310
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: anti-CRISPR protein AcrF1 fused with C-terminal MBP tag
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,50212
Polymers49,7731
Non-polymers1,72911
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)87.389, 63.245, 96.733
Angle α, β, γ (deg.)90.00, 93.21, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein anti-CRISPR protein AcrF1 fused with C-terminal MBP tag / MBP / MMBP / Maltodextrin-binding protein


Mass: 49773.141 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage JBD30 (virus), (gene. exp.) Escherichia coli O157:H7 (bacteria)
Gene: JBD30_035, malE, Z5632, ECs5017 / Plasmid: pRSFDuet-SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: L7P7M1, UniProt: P0AEY0
#2: Polysaccharide alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose / alpha-maltotetraose


Type: oligosaccharide, Oligosaccharide / Class: Substrate analog / Mass: 666.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltotetraose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,4,3/[a2122h-1a_1-5]/1-1-1-1/a4-b1_b4-c1_c4-d1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}}}LINUCSPDB-CARE

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Non-polymers , 5 types, 272 molecules

#3: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#4: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H14O4
#5: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C2H6O2
#6: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C4H10O3
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 253 / 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: 2.69 Å3/Da / Density % sol: 54.27 %
Crystal growTemperature: 293 K / Method: evaporation / pH: 6.2
Details: 0.1 M MES (pH 6.2), 2.5% PEG 3000 (v/v), and 42% PEG400 (v/v)

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Aug 11, 2016 / Details: LR-Design detector positioner
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.3→50 Å / Num. obs: 47647 / % possible obs: 98.7 % / Redundancy: 4.1 % / Biso Wilson estimate: 26.44 Å2 / Rmerge(I) obs: 0.125 / Rpim(I) all: 0.066 / Rrim(I) all: 0.142 / Χ2: 1.03 / Net I/σ(I): 5.3 / Num. measured all: 195221
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.3-2.383.30.41146680.840.2340.4760.84397.2
2.38-2.483.70.35847040.8710.1960.4110.8998.6
2.48-2.594.20.31647820.9120.1610.3570.93599.5
2.59-2.734.30.26447430.9270.1360.2990.97599
2.73-2.94.10.21347530.9550.1110.2411.0798.6
2.9-3.124.10.1747590.9640.0890.1931.12199.2
3.12-3.444.40.13247750.9810.0670.1491.21399.1
3.44-3.934.10.10247540.9830.0550.1171.27398.4
3.93-4.954.40.08348360.9890.0430.0941.07399.3
4.95-504.10.0748730.9910.0370.080.83697.9

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
HKL-2000data scaling
PDB_EXTRACT3.22data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4EXK

4exk


Resolution: 2.265→45.871 Å / SU ML: 0.24 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 21.59 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.223 2310 4.85 %
Rwork0.1648 --
obs0.1676 47627 96.42 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.265→45.871 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7014 0 221 253 7488
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0087386
X-RAY DIFFRACTIONf_angle_d0.9359975
X-RAY DIFFRACTIONf_dihedral_angle_d22.8762897
X-RAY DIFFRACTIONf_chiral_restr0.1591088
X-RAY DIFFRACTIONf_plane_restr0.0061268
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.2649-2.31110.2442830.21431619X-RAY DIFFRACTION59
2.3111-2.36130.31711390.19262698X-RAY DIFFRACTION98
2.3613-2.41630.23411340.1852648X-RAY DIFFRACTION98
2.4163-2.47670.26541390.18422725X-RAY DIFFRACTION99
2.4767-2.54360.27151350.17312734X-RAY DIFFRACTION99
2.5436-2.61850.23151240.17272756X-RAY DIFFRACTION99
2.6185-2.7030.25821410.16542710X-RAY DIFFRACTION99
2.703-2.79960.21481350.16592721X-RAY DIFFRACTION99
2.7996-2.91170.2641120.17742740X-RAY DIFFRACTION99
2.9117-3.04420.21991440.18312740X-RAY DIFFRACTION99
3.0442-3.20460.24011600.17452720X-RAY DIFFRACTION99
3.2046-3.40530.22981480.17112714X-RAY DIFFRACTION99
3.4053-3.66820.19431450.15732721X-RAY DIFFRACTION99
3.6682-4.03710.20661340.14192743X-RAY DIFFRACTION98
4.0371-4.62080.20121390.13592785X-RAY DIFFRACTION99
4.6208-5.81990.1941330.16062752X-RAY DIFFRACTION98
5.8199-45.87990.20841650.16982791X-RAY DIFFRACTION98
Refinement TLS params.Method: refined / Origin x: -70.2402 Å / Origin y: -58.7448 Å / Origin z: 25.1715 Å
111213212223313233
T0.1498 Å20.002 Å20.0049 Å2-0.1703 Å2-0.0349 Å2--0.1812 Å2
L0.0296 °20.0091 °2-0.0604 °2-0.2057 °2-0.1941 °2--0.7495 °2
S0.0108 Å °-0.0193 Å °0.0104 Å °-0.0016 Å °-0.0125 Å °-0.0552 Å °0.0092 Å °0.1434 Å °0.0044 Å °
Refinement TLS groupSelection details: all

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