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- PDB-6pew: CryoEM Plasmodium falciparum glutamine synthetase -

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Basic information

Entry
Database: PDB / ID: 6pew
TitleCryoEM Plasmodium falciparum glutamine synthetase
ComponentsGlutamine synthetase
KeywordsLIGASE / glutamine synthetase
Function / homology
Function and homology information


glutamine biosynthetic process / glutamine synthetase activity
Similarity search - Function
Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase, N-terminal domain / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, catalytic domain / Glutamine synthetase, catalytic domain / Glutamine synthetase/guanido kinase, catalytic domain
Similarity search - Domain/homology
Glutamine synthetase / Glutamine synthetase, type I
Similarity search - Component
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHo, C.M. / Lai, M. / Zhou, Z.H.
Funding support United States, 10items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01GM071940 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)AI094386 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)DE025567 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10RR23057 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10OD018111 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)U24GM116792 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32 AI007323 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)K99/R00 HL133453 United States
CitationJournal: Nat Methods / Year: 2020
Title: Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu.
Authors: Chi-Min Ho / Xiaorun Li / Mason Lai / Thomas C Terwilliger / Josh R Beck / James Wohlschlegel / Daniel E Goldberg / Anthony W P Fitzpatrick / Z Hong Zhou /
Abstract: X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up ...X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (cryoEM) maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria-causing parasite Plasmodium falciparum, an organism that has resisted conventional structural-biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.
History
DepositionJun 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 11, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 15, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
D: Glutamine synthetase
G: Glutamine synthetase
H: Glutamine synthetase
I: Glutamine synthetase
J: Glutamine synthetase
K: Glutamine synthetase
L: Glutamine synthetase
A: Glutamine synthetase
B: Glutamine synthetase
C: Glutamine synthetase
E: Glutamine synthetase
F: Glutamine synthetase


Theoretical massNumber of molelcules
Total (without water)759,71512
Polymers759,71512
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Glutamine synthetase /


Mass: 63309.551 Da / Num. of mol.: 12 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate NF54) (eukaryote)
Strain: isolate NF54 / References: UniProt: A0A2I0BT46, UniProt: W7JM41*PLUS

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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: Plasmodium falciparum glutamine synthetase / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Plasmodium falciparum (isolate NF54) (eukaryote)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated defocus min: 1500 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
4cryoSPARC1CTF correction
10cryoSPARC1initial Euler assignment
11cryoSPARC1final Euler assignment
13cryoSPARC13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8350 / Symmetry type: POINT

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