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Basic information

Entry
Database: PDB / ID: 6vrs
TitleSingle particle reconstruction of glucose isomerase from Streptomyces rubiginosus based on data acquired in the presence of substantial aberrations
Componentsxylose isomerase
KeywordsISOMERASE / glucose isomerase
Function / homology
Function and homology information


xylose isomerase / D-xylose metabolic process / xylose isomerase activity / magnesium ion binding / identical protein binding / cytoplasm
Similarity search - Function
Xylose isomerase, actinobacteria / Xylose isomerase / Xylose isomerase family profile. / Divalent-metal-dependent TIM barrel enzymes / Xylose isomerase-like, TIM barrel domain / Xylose isomerase-like TIM barrel / Xylose isomerase-like superfamily / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
: / Xylose isomerase
Similarity search - Component
Biological speciesStreptomyces rubiginosus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBromberg, R. / Guo, Y. / Borek, D. / Otwinowski, Z.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117080 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM118619 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R21GM126406 United States
Department of Energy (DOE, United States)DE-SC0019600 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)HHSN272201700060C United States
Citation
Journal: IUCrJ / Year: 2020
Title: High-resolution cryo-EM reconstructions in the presence of substantial aberrations.
Authors: Raquel Bromberg / Yirui Guo / Dominika Borek / Zbyszek Otwinowski /
Abstract: Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula ...Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula quantifying information loss owing to their presence is inferred that explains why Fourier-shell coefficient-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. The analysis is validated with reference-based aberration refinement for two cryo-EM SPR data sets acquired with a 200 kV microscope in the presence of coma exceeding 40 µm, and 2.3 and 2.7 Å reconstructions for 144 and 173 kDa particles, respectively, were obtained. The results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher order aberrations, and address inconsistent guidelines regarding the level of aberrations that is acceptable in cryo-EM SPR experiments.
#1: Journal: Biorxiv / Year: 2020
Title: High-resolution cryo-EM reconstructions in the presence of substantial aberrations
Authors: Bromberg, R. / Guo, Y. / Borek, D. / Otwinowski, Z.
History
DepositionFeb 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1May 13, 2020Group: Database references / Category: citation / citation_author
Revision 1.2Mar 6, 2024Group: 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 / citation / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ncs_dom_lim
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _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 / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_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

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-21371
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: xylose isomerase
B: xylose isomerase
C: xylose isomerase
D: xylose isomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)173,57312
Polymers173,1334
Non-polymers4408
Water28816
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:

Component-ID: 1 / Beg auth comp-ID: ASN / Beg label comp-ID: ASN / End auth comp-ID: GLY / End label comp-ID: GLY / Refine code: 1 / Auth seq-ID: 2 - 388 / Label seq-ID: 2 - 388

Dom-IDEns-IDAuth asym-IDLabel asym-ID
11AA
21BB
12AA
22CC
13AA
23DD
14BB
24CC
15BB
25DD
16CC
26DD

NCS ensembles :
ID
1
2
3
4
5
6

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-1, -0.000246, -0.000207), (0.000246, -1, -3.9E-5), (-0.000207, -3.9E-5, 1)91.02138, 85.53458, 0.01184
3given(1), (1), (1)
4given(-1, 0.000169, 0.000241), (0.000169, 1, -0.000113), (-0.000241, -0.000113, -1)90.98003, 0.00166, 101.93268
5given(1), (1), (1)
6given(1, -3.7E-5, -1.3E-5), (-3.7E-5, -1, -1.7E-5), (-1.3E-5, 1.7E-5, -1)0.00259, 85.54288, 101.91992
7given(1), (1), (1)
8given(1, 7.7E-5, -3.4E-5), (7.7E-5, -1, 0.000152), (-3.4E-5, -0.000152, -1)-0.00074, 85.52891, 101.9242
9given(1), (1), (1)
10given(-1, -0.000213, 0.000219), (-0.000213, 1, 5.8E-5), (-0.000219, 5.8E-5, -1)90.99798, 0.01026, 101.92677
11given(1), (1), (1)
12given(-1, -0.000136, 0.000253), (0.000136, -1, 9.4E-5), (0.000253, 9.4E-5, 1)90.99215, 85.52562, -0.0187

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Components

#1: Protein
xylose isomerase / glucose isomerase


Mass: 43283.297 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Streptomyces rubiginosus (bacteria) / References: UniProt: P24300, xylose isomerase
#2: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: glucose isomerase / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.1729 MDa / Experimental value: YES
Source (natural)Organism: Streptomyces rubiginosus (bacteria)
Buffer solutionpH: 7.5
Buffer componentConc.: 20 mM / Name: HEPES
SpecimenConc.: 40 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Details: The goal of the experiment was to show that it is possible to perform high resolution reconstruction in the presence of higher order aberrations.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Residual tilt: 5.3 mradians
Image recordingAverage exposure time: 100 sec. / Electron dose: 120 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 202
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 200 / Used frames/image: 1-200

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Processing

SoftwareName: REFMAC / Version: 5.8.0257 / Classification: refinement
EM software
IDNameCategory
1cisTEMparticle selection
2SerialEMimage acquisition
4cisTEMCTF correction
7MOLREPmodel fitting
9cisTEMinitial Euler assignment
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 114522
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61909 / Algorithm: BACK PROJECTION / Details: We used the approach implemented in cisTEM. / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL / Target criteria: REFMAC / Details: COOT was crucial as well.
Atomic model buildingPDB-ID: 5VR0

5vr0


Accession code: 5VR0 / Source name: PDB / Type: experimental model
RefinementResolution: 2.7→100.1 Å / Cor.coef. Fo:Fc: 0.972 / SU B: 11.452 / SU ML: 0.218 / ESU R: 0.417
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.245 --
obs0.245 79487 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 80.52 Å2
Baniso -1Baniso -2Baniso -3
1-0.62 Å2-0 Å20.01 Å2
2--1.57 Å2-0 Å2
3----2.19 Å2
Refinement stepCycle: 1 / Total: 12208
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01212472
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.6021.63916884
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.40551544
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.24820.784816
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.633151960
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.59515140
ELECTRON MICROSCOPYr_chiral_restr0.1150.21508
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.0210140
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it5.4177.4136188
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it8.60811.1477728
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it7.0818.3746284
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined16.00146781
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Number: 3046 / Refine-ID: ELECTRON MICROSCOPY / Type: tight thermal / Weight position: 0.87

Ens-IDDom-IDAuth asym-IDRms dev position (Å)
11A1.05
22B1.08
33C0.35
44D0.38
55A1.02
66B1.04
LS refinement shellResolution: 2.7→2.77 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.745 5855 -
obs--100 %

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