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- PDB-8pvf: Structure of GAPDH determined by cryoEM at 100 keV -

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Basic information

Entry
Database: PDB / ID: 8pvf
TitleStructure of GAPDH determined by cryoEM at 100 keV
ComponentsGlyceraldehyde-3-phosphate dehydrogenase
KeywordsOXIDOREDUCTASE / GAPDH
Function / homology
Function and homology information


glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity / glycolytic process / glucose metabolic process / NAD binding / NADP binding / identical protein binding / cytosol
Similarity search - Function
Glyceraldehyde 3-phosphate dehydrogenase, active site / Glyceraldehyde 3-phosphate dehydrogenase active site. / Glyceraldehyde-3-phosphate dehydrogenase, type I / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD(P) binding domain / Glyceraldehyde 3-phosphate dehydrogenase, catalytic domain / Glyceraldehyde/Erythrose phosphate dehydrogenase family / Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Glyceraldehyde-3-phosphate dehydrogenase
Similarity search - Component
Biological speciesCryptosporidium parvum (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsMcMullan, G. / Naydenova, K. / Mihaylov, D. / Peet, M.J. / Wilson, H. / Yamashita, K. / Dickerson, J.L. / Chen, S. / Cannone, G. / Lee, Y. ...McMullan, G. / Naydenova, K. / Mihaylov, D. / Peet, M.J. / Wilson, H. / Yamashita, K. / Dickerson, J.L. / Chen, S. / Cannone, G. / Lee, Y. / Hutchings, K.A. / Gittins, O. / Sobhy, M. / Wells, T. / El-Gomati, M.M. / Dalby, J. / Meffert, M. / Schulze-Briese, C. / Henderson, R. / Russo, C.J.
Funding support United Kingdom, 6items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC UP 120117 United Kingdom
Medical Research Council (MRC, United Kingdom)MC U105184322 United Kingdom
Wellcome Trust220526/B/20/Z United Kingdom
Engineering and Physical Sciences Research CouncilR122522 United Kingdom
Innovate UK103806 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T003677/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structure determination by cryoEM at 100 keV.
Authors: Greg McMullan / Katerina Naydenova / Daniel Mihaylov / Keitaro Yamashita / Mathew J Peet / Hugh Wilson / Joshua L Dickerson / Shaoxia Chen / Giuseppe Cannone / Yang Lee / Katherine A ...Authors: Greg McMullan / Katerina Naydenova / Daniel Mihaylov / Keitaro Yamashita / Mathew J Peet / Hugh Wilson / Joshua L Dickerson / Shaoxia Chen / Giuseppe Cannone / Yang Lee / Katherine A Hutchings / Olivia Gittins / Mohamed A Sobhy / Torquil Wells / Mohamed M El-Gomati / Jason Dalby / Matthias Meffert / Clemens Schulze-Briese / Richard Henderson / Christopher J Russo /
Abstract: Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose- ...Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.
History
DepositionJul 17, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glyceraldehyde-3-phosphate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,7232
Polymers38,0591
Non-polymers6631
Water00
1
A: Glyceraldehyde-3-phosphate dehydrogenase
hetero molecules

A: Glyceraldehyde-3-phosphate dehydrogenase
hetero molecules

A: Glyceraldehyde-3-phosphate dehydrogenase
hetero molecules

A: Glyceraldehyde-3-phosphate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)154,8928
Polymers152,2384
Non-polymers2,6544
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-1, -1.2246468E-16), (1.2246468E-16, -1), (1)215.424, 215.424
3generate(1), (-1, -1.2246468E-16), (1.2246468E-16, -1)215.424, 215.424
4generate(-1, 1.2246468E-16), (1), (-1.2246468E-16, -1)215.424, 215.424

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Components

#1: Protein Glyceraldehyde-3-phosphate dehydrogenase


Mass: 38059.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cryptosporidium parvum (eukaryote) / Gene: cgd6_3790, 1MB.519, CPATCC_002941 / Production host: Escherichia coli (E. coli)
References: UniProt: Q7YYQ9, glyceraldehyde-3-phosphate dehydrogenase (phosphorylating)
#2: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
Has ligand of interestN

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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: Glyceraldehyde 3-phosphate dehydrogenase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Cryptosporidium parvum (eukaryote)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R0./1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL 1400/HR + YPS FEG
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 100 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: DECTRIS SINGLA (1k x 1k)

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Processing

EM softwareName: Servalcat / Version: 0.4.27 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19411 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
Atomic model buildingPDB-ID: 3cps

3cps


Accession code: 3cps / Source name: PDB / Type: experimental model

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