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- PDB-6vhc: 1.4A damaged structure of GSNQNNF used to determine initial phase... -

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Basic information

Entry
Database: PDB / ID: 6vhc
Title1.4A damaged structure of GSNQNNF used to determine initial phases from radiation damage
ComponentsGSNQNNF
KeywordsPROTEIN FIBRIL / MicroED / damage / phasing / RIP
Function / homologyACETATE ION
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.4 Å
AuthorsMartynowycz, M.W. / Hattne, J. / Gonen, T.
CitationJournal: Structure / Year: 2020
Title: Experimental Phasing of MicroED Data Using Radiation Damage.
Authors: Michael W Martynowycz / Johan Hattne / Tamir Gonen /
Abstract: We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present ...We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present an example of an experimentally phased structure using only MicroED data. The structure of a seven-residue peptide is solved starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous-rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a "low-dose dataset." The data from the second pass were similarly merged to form a "damaged dataset." Differences between these two datasets were used to identify a single heavy-atom site from a Patterson difference map, and initial phases were generated. Finally, the structure was completed by iterative cycles of modeling and refinement.
History
DepositionJan 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 22, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
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 / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_conn_angle / 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 / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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_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_conn.ptnr2_symmetry

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Assembly

Deposited unit
A: GSNQNNF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)9043
Polymers7801
Non-polymers1242
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)4.880, 14.170, 17.620
Angle α, β, γ (deg.)83.600, 84.980, 83.310
Int Tables number1
Space group name H-MP1

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Components

#1: Protein/peptide GSNQNNF


Mass: 779.756 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / 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: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Synthetic proto-filament / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.000899141 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
10.1 MMESC6H13NO4S1
210 %MPDC6H14O21
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Hanging drop.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 30 %

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 2.1 sec. / Electron dose: 0.00588 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of diffraction images: 736 / Num. of grids imaged: 1 / Num. of real images: 736 / Details: Images collected as a movies.
Image scansSampling size: 31.2 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 730 mm
EM diffraction shellResolution: 1.4→13.97 Å / Fourier space coverage: 78.1 % / Multiplicity: 8.7 / Num. of structure factors: 722 / Phase residual: 26 °
EM diffraction statsDetails: Model from the low-dose set refined against the damage dataset without any changes.
Fourier space coverage: 78.1 % / High resolution: 1.4 Å / Num. of intensities measured: 6314 / Num. of structure factors: 722 / Phase error: 26 ° / Phase residual: 26 ° / Phase error rejection criteria: 0 / Rmerge: 0.199 / Rsym: 0.21
ReflectionHighest resolution: 1.4 Å / Num. obs: 722 / % possible obs: 78.1 % / Redundancy: 8.745 % / Biso Wilson estimate: 10.108 Å2 / CC1/2: 0.982 / Rmerge(I) obs: 0.199 / Rrim(I) all: 0.21 / Χ2: 0.921 / Net I/σ(I): 8.67 / Num. measured all: 6314 / Scaling rejects: 1
Reflection shell

Diffraction-ID: 1

Resolution (Å)Highest resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
1.4-1.519.3830.2726.614451951540.9630.28779
1.51-1.669.110.213813211831450.9720.22479.2
1.66-1.857.6130.2128.158071421060.9420.22674.6
1.85-2.148.4120.1989.529591461140.9790.20978.1
2.14-2.629.2220.18510.37913125990.9860.19479.2
2.62-3.77.7730.18610.0951383660.970.19779.5
3.79.3680.19211.5235650380.9870.20276

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Processing

Software
NameVersionClassificationNB
SHELXrefinement
XSCALEdata scaling
PDB_EXTRACT3.25data extraction
EM softwareName: EM-Menu / Version: 4.0.9.75 / Category: image acquisition
Image processingDetails: Rolling shutter and binned by 2.
EM 3D crystal entity∠α: 83.7 ° / ∠β: 85.43 ° / ∠γ: 82.96 ° / A: 4.85 Å / B: 14.15 Å / C: 17.43 Å / Space group name: 1 / Space group num: 1
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 5 / Protocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 6CLJ
Pdb chain-ID: A / Accession code: 6CLJ / Pdb chain residue range: 1-7 / Source name: PDB / Type: experimental model
RefinementResolution: 1.4→13.97 Å / Cross valid method: THROUGHOUT
RfactorNum. reflection% reflectionSelection details
Rfree0.322 --Rcomplete random
Rwork0.243 ---
obs-722 78.1 %-
Displacement parametersBiso max: 18.27 Å2 / Biso mean: 4.5087 Å2 / Biso min: 0.08 Å2

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