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- PDB-6mxf: MicroED structure of thiostrepton at 1.9 A resolution -

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Entry
Database: PDB / ID: 6mxf
TitleMicroED structure of thiostrepton at 1.9 A resolution
ComponentsThiostrepton
KeywordsANTIBIOTIC
Function / homologyThiazolylpeptide-type bacteriocin precursor / defense response to bacterium / extracellular region / THIOSTREPTON / Thiostrepton
Function and homology information
Biological speciesStreptomyces azureus (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 1.91 Å
AuthorsJones, C.G. / Martynowycz, M.W. / Hattne, J. / Fulton, T. / Stoltz, B.M. / Rodriguez, J.A. / Nelson, H.M. / Gonen, T.
Funding support United States, 5items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DGE-1650604 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM080269 United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128867 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128936 United States
CitationJournal: ACS Cent Sci / Year: 2018
Title: The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination.
Authors: Christopher G Jones / Michael W Martynowycz / Johan Hattne / Tyler J Fulton / Brian M Stoltz / Jose A Rodriguez / Hosea M Nelson / Tamir Gonen /
Abstract: In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful ...In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule's structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10 g) resulting in atomic resolution (<1 Å) crystal structures in minutes.
History
DepositionOct 30, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 21, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 2, 2019Group: Data collection / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / diffrn_radiation_wavelength / diffrn_source / struct_site / struct_site_gen
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 6, 2019Group: Data collection / Database references / Category: struct_ref / Item: _struct_ref.db_name / _struct_ref.pdbx_db_accession
Revision 2.0Nov 27, 2019Group: Author supporting evidence / Polymer sequence / Category: entity_poly / pdbx_audit_support
Item: _entity_poly.pdbx_seq_one_letter_code_can / _pdbx_audit_support.funding_organization
Revision 2.1Oct 11, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id
Revision 2.2Nov 15, 2023Group: Data collection / Derived calculations / Category: chem_comp_atom / chem_comp_bond / struct_conn
Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2 / _struct_conn.pdbx_leaving_atom_flag

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Assembly

Deposited unit
A: Thiostrepton


Theoretical massNumber of molelcules
Total (without water)1,8061
Polymers1,8061
Non-polymers00
Water362
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area400 Å2
ΔGint1 kcal/mol
Surface area1690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)26.219, 26.219, 27.534
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein/peptide Thiostrepton / /


Type: Thiopeptide / Class: Antibiotic / Mass: 1805.985 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Thiostrepton is a hetrocyclic thiopeptide belonging to the thiocillin family, consisting of four thiazole, one thiozoline and one piperideine rings. A modified quinoline linked to main-chain ...Details: Thiostrepton is a hetrocyclic thiopeptide belonging to the thiocillin family, consisting of four thiazole, one thiozoline and one piperideine rings. A modified quinoline linked to main-chain residue 1 and side-chain of residue 12. Post translational maturation of thiazole and oxazole containing antibiotics involves the enzymic condensation of a Cys or Ser with the alpha-carbonyl of the preceding amino acid to form a thioether or ether bond, then dehydration to form a double bond with the alpha-amino nitrogen. Thiazoline or oxazoline ring are dehydrogenated to form thiazole or oxazole rings. the pyridinyl involves the cross-linking of a Ser and a Cys-Ser pair usually separated by 7 or 8 residues along the peptide chain. The Ser residues are dehydrated to didehydroalanines, then bonded between their beta carbons. The alpha carbonyl of the Cys condenses with alpha carbon of the first Ser to form a pyridinyl ring. The ring may be mutiply dehydrogenated to form a pyridine ring with loss of the amino nitrogen of the first Ser. The amidation of Ser-17 probably does not occur by the same mechanism, oxidative cleavage of glycine, as in eukaryotes.
Source: (synth.) Streptomyces azureus (bacteria) / References: THIOSTREPTON, UniProt: P0C8P8*PLUS
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
Compound detailsTHIOSTREPTON IS A MEMBER OF A SULPHUR-RICH HETEROCYCLIC PEPTIDES CLASS. ALL SHARE A MACROCYLIC ...THIOSTREPTON IS A MEMBER OF A SULPHUR-RICH HETEROCYCLIC PEPTIDES CLASS. ALL SHARE A MACROCYLIC CORE, CONSISTING OF A NITROGEN CONTAINING, SIX-MEMBERED RING CENTRAL TO DEHYDROAMINO ACIDS AND A SUBSET OF FIVE MEMBER RING STRUCTURES INCLUDING THIAZOLES, THIAZOLINES AND OXAZOLES. HERE, THIOSTREPTON IS REPRESENTED BY THE SEQUENCE (SEQRES)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Thiostrepton / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Molecular weightValue: .001619538000 MDa / Experimental value: NO
Source (natural)Organism: Streptomyces azureus (bacteria)
EM crystal formationDetails: Powder
Buffer solutionpH: 7 / Details: Powder
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Powder
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 298 K / Details: Hand-plunged

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.21 sec. / Electron dose: 0.09 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 214 / Num. of grids imaged: 1 / Details: FEI CetaD
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 960 mm
EM diffraction shellResolution: 1.91→2.13 Å / Fourier space coverage: 40.3 % / Multiplicity: 4.9 / Num. of structure factors: 92 / Phase residual: 29.19 °
EM diffraction statsFourier space coverage: 78.6 % / High resolution: 1.91 Å / Num. of intensities measured: 5578 / Num. of structure factors: 686 / Phase error: 26.93 ° / Phase residual: 26.93 ° / Phase error rejection criteria: 0 / Rmerge: 0.236 / Rsym: 0.236
Diffraction sourceWavelength: 0.0251 Å
DetectorDate: Oct 3, 2018
Radiation wavelengthWavelength: 0.0251 Å / Relative weight: 1
ReflectionResolution: 1.9→18.99 Å / Num. obs: 686 / % possible obs: 78.6 % / Redundancy: 8.1 % / CC1/2: 0.985 / Rmerge(I) obs: 0.236 / Rpim(I) all: 0.084 / Rrim(I) all: 0.251 / Net I/σ(I): 5.1
Reflection shellResolution: 1.9→2.13 Å / Redundancy: 4.9 % / Rmerge(I) obs: 0.32 / Mean I/σ(I) obs: 3.4 / Num. unique all: 93 / CC1/2: 0.813 / % possible all: 40.3

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
MOSFLM7.2.2data reduction
Aimless0.7.3data scaling
MOLREP11.6.04phasing
EM software
IDNameVersionCategory
6MOLREP11.6.04model fitting
8MOLREP11.6.04molecular replacement
10POINTLESS1.11.14symmetry determination
11AIMLESS0.7.3crystallography merging
13REFMAC5.8.0232model refinement
Image processingDetails: FEI Ceta
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 26.219 Å / B: 26.219 Å / C: 27.534 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.91 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 2.6 / Protocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 1E9W

1e9w


Pdb chain-ID: A / Accession code: 1E9W / Pdb chain residue range: 0-18 / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1e9w

1e9w


Resolution: 1.91→18.99 Å / Cor.coef. Fo:Fc: 0.931 / Cor.coef. Fo:Fc free: 0.94 / SU B: 3.282 / SU ML: 0.098 / Cross valid method: THROUGHOUT / ESU R: 0.352 / ESU R Free: 0.202 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.21798 57 8.4 %RANDOM
Rwork0.19046 ---
obs0.19276 620 78.18 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 6.811 Å2
Baniso -1Baniso -2Baniso -3
1-0.18 Å2-0 Å2-0 Å2
2--0.18 Å2-0 Å2
3----0.37 Å2
Refinement stepCycle: 1 / Total: 116
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0150.017123
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0250.018107
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg2.762.241120
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.3742.283213
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg3.62353
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg7.901154
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0620.215
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.010.02135
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0030.0249
ELECTRON CRYSTALLOGRAPHYr_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_nbd_other
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined
ELECTRON CRYSTALLOGRAPHYr_nbtor_other
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_other
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_mcbond_it0.6250.79862
ELECTRON CRYSTALLOGRAPHYr_mcbond_other0.620.8163
ELECTRON CRYSTALLOGRAPHYr_mcangle_it0.8931.19437
ELECTRON CRYSTALLOGRAPHYr_mcangle_other0.8811.22338
ELECTRON CRYSTALLOGRAPHYr_scbond_it0.6090.74360
ELECTRON CRYSTALLOGRAPHYr_scbond_other0.6040.75461
ELECTRON CRYSTALLOGRAPHYr_scangle_it
ELECTRON CRYSTALLOGRAPHYr_scangle_other0.8921.16184
ELECTRON CRYSTALLOGRAPHYr_long_range_B_refined1.54610.96101
ELECTRON CRYSTALLOGRAPHYr_long_range_B_other1.53910.959102
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr
ELECTRON CRYSTALLOGRAPHYr_sphericity_free
ELECTRON CRYSTALLOGRAPHYr_sphericity_bonded
LS refinement shellResolution: 1.915→1.964 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.182 2 -
obs--3.33 %

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