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- PDB-8pve: Structure of AHIR determined by cryoEM at 100 keV -

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Basic information

Entry
Database: PDB / ID: 8pve
TitleStructure of AHIR determined by cryoEM at 100 keV
ComponentsKetol-acid reductoisomerase (NADP(+))
KeywordsOXIDOREDUCTASE
Function / homology
Function and homology information


ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / valine biosynthetic process / isoleucine biosynthetic process / NADP binding / magnesium ion binding
Similarity search - Function
Ketol-acid reductoisomerase, prokaryotic / Ketol-acid reductoisomerase, C-terminal / Ketol-acid reductoisomerase / Ketol-acid reductoisomerase, N-terminal / Acetohydroxy acid isomeroreductase, catalytic domain / Acetohydroxy acid isomeroreductase, NADPH-binding domain / KARI N-terminal domain profile. / KARI C-terminal domain profile. / 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Ketol-acid reductoisomerase (NADP(+))
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 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: Ketol-acid reductoisomerase (NADP(+))


Theoretical massNumber of molelcules
Total (without water)37,9941
Polymers37,9941
Non-polymers00
Water00
1
A: Ketol-acid reductoisomerase (NADP(+))
x 12


Theoretical massNumber of molelcules
Total (without water)455,93112
Polymers455,93112
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation11
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-0.5, -0.866025404), (0.866025404, -0.5), (1)413.143526, 110.701474
3generate(-0.5, 0.866025404), (-0.866025404, -0.5), (1)110.701474, 413.143526
4generate(-0.5, 0.288675132, 0.816496582), (-0.288675132, 0.833333336, -0.471404518), (-0.816496582, -0.471404518, -0.333333336)68.9429411, 161.823808, 457.706851
5generate(-0.5, -0.288675132, -0.816496582), (0.288675132, 0.833333336, -0.471404518), (0.816496582, -0.471404518, -0.333333336)454.902059, 61.0097912, 172.56175
6generate(0.500000004, -0.866025401, -1.1063965E-8), (-0.288675125, -0.166666675, 0.942809043), (-0.816496582, -0.471404522, -0.333333329)238.528527, 89.4959074, 457.706851
7generate(0.500000004, -0.288675125, -0.816496582), (-0.866025401, -0.166666675, -0.471404522), (-1.10639654E-8, 0.942809043, -0.333333329)280.287057, 437.252828, 68.1914002
8generate(0.49999999, 0.288675134, 0.816496588), (0.86602541, -0.16666666, -0.471404512), (-1.06669434E-9, 0.942809043, -0.333333329)-105.672058, 134.810771, 68.1913984
9generate(0.49999999, 0.86602541, -1.06669401E-9), (0.288675134, -0.16666666, 0.942809043), (0.816496588, -0.471404512, -0.333333329)-63.9135249, -11.3181106, 172.561746
10generate(-1.86513027E-9, -0.577350272, 0.816496579), (-0.577350272, -0.666666662, -0.471404523), (0.816496579, -0.471404523, -0.333333336)132.856468, 474.153318, 172.561751
11generate(5.48605983E-9, 0.577350268, -0.816496582), (0.577350268, -0.66666667, -0.471404518), (-0.816496582, -0.471404518, -0.333333336)216.373533, 272.525283, 457.706851
12generate(-1, -1.56767344E-8, -1.10851251E-8), (-1.56767343E-8, 0.333333329, 0.942809043), (-1.10851253E-8, 0.942809043, -0.333333329)349.230005, -48.2185976, 68.1914002

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Components

#1: Protein Ketol-acid reductoisomerase (NADP(+)) / KARI / Acetohydroxy-acid isomeroreductase / AHIR / Alpha-keto-beta-hydroxylacyl reductoisomerase / ...KARI / Acetohydroxy-acid isomeroreductase / AHIR / Alpha-keto-beta-hydroxylacyl reductoisomerase / Ketol-acid reductoisomerase type 1 / Ketol-acid reductoisomerase type I


Mass: 37994.246 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Gene: ilvC, TT_C0850 / Production host: Escherichia coli (E. coli)
References: UniProt: Q72JC8, ketol-acid reductoisomerase (NADP+)

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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: Acetohydroxy acid isomeroreductase / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Thermus thermophilus (bacteria)
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: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: 80 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: T (tetrahedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15220 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL

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