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- PDB-8pvj: Structure of lumazine synthase determined by cryoEM at 100 keV -

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Basic information

Entry
Database: PDB / ID: 8pvj
TitleStructure of lumazine synthase determined by cryoEM at 100 keV
Components6,7-dimethyl-8-ribityllumazine synthaseLumazine synthase
KeywordsTRANSFERASE / LUMAZINE SYNTHASE
Function / homology
Function and homology information


6,7-dimethyl-8-ribityllumazine synthase / 6,7-dimethyl-8-ribityllumazine synthase activity / riboflavin synthase complex / riboflavin biosynthetic process / cytosol
Similarity search - Function
Lumazine synthase / Lumazine/riboflavin synthase / Lumazine/riboflavin synthase superfamily / 6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Domain/homology
6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 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: 6,7-dimethyl-8-ribityllumazine synthase


Theoretical massNumber of molelcules
Total (without water)17,7691
Polymers17,7691
Non-polymers00
Water0
1
A: 6,7-dimethyl-8-ribityllumazine synthase
x 60


Theoretical massNumber of molelcules
Total (without water)1,066,16160
Polymers1,066,16160
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(0.309016994, -0.499999578, 0.809017255), (0.499999578, 0.809017316, 0.309016833), (-0.809017255, 0.309016833, 0.499999678)50.633324, -81.926551, 132.560099
3generate(-0.809016994, -0.309016734, 0.500000161), (0.309016734, 0.500000843, 0.809016573), (-0.500000161, 0.809016573, -0.309017837)214.48653, -81.9266069, 132.560189
4generate(-0.809016994, 0.309016734, -0.500000161), (-0.309016734, 0.500000843, 0.809016573), (0.500000161, 0.809016573, -0.309017837)265.120056, -9.04142101E-5, 0.000146293435
5generate(0.309016994, 0.499999578, -0.809017255), (-0.499999578, 0.809017316, 0.309016833), (0.809017255, 0.309016833, 0.499999678)132.56009, 50.6333372, -81.9265561
6generate(0.499999678, -0.809017255, 0.309016833), (0.809017255, 0.309016994, -0.499999578), (0.309016833, 0.499999578, 0.809017316)132.560099, 50.633324, -81.926551
7generate(-0.309017837, -0.500000161, 0.809016573), (0.500000161, -0.809016994, -0.309016734), (0.809016573, 0.309016734, 0.500000843)132.560189, 214.48653, -81.9266069
8generate(-0.309017837, 0.500000161, 0.809016573), (-0.500000161, -0.809016994, 0.309016734), (0.809016573, -0.309016734, 0.500000843)0.000146293435, 265.120056, -9.04142101E-5
9generate(0.499999678, 0.809017255, 0.309016833), (-0.809017255, 0.309016994, 0.499999578), (0.309016833, -0.499999578, 0.809017316)-81.9265561, 132.56009, 50.6333372
10generate(1.68874961E-7, 5.36935615E-8, 1), (1, -1.15181379E-7, -1.68874954E-7), (1.1518137E-7, 1, -5.36935808E-8)-2.95036813E-5, 3.76545104E-5, -8.15082032E-6
11generate(1.6887496E-7, 1, 1.15181371E-7), (5.3693562E-8, -1.1518138E-7, 1), (1, -1.68874954E-7, -5.36935812E-8)-3.76545045E-5, 8.15082618E-6, 2.95036871E-5
12generate(-0.5, -0.809017081, 0.309016767), (0.80901666, -0.309016593, 0.500000789), (-0.309017871, 0.500000107, 0.809016593)265.120042, -0.00011343249, 0.000155088677
13generate(-0.5, 0.80901666, -0.309017871), (-0.809017081, -0.309016593, 0.500000107), (0.309016767, 0.500000789, 0.809016593)132.560161, 214.48653, -81.9266069
14generate(0.309016994, 0.499999318, 0.809017416), (0.499999318, -0.809017515, 0.309016734), (0.809017416, 0.309016734, -0.499999479)-81.926551, 132.560194, 50.6333241
15generate(-1, -3.73475425E-13, -1.2032E-6), (-3.73230495E-13, -1, 6.206E-7), (-1.2032E-6, 6.206E-7, 1)265.120159, 265.119918, 7.72295774E-5
16generate(0.809016279, -0.309018627, 0.500000148), (0.309016519, -0.5, -0.809017176), (0.500001451, 0.809016371, -0.309016279)0.000291554105, 265.120087, -0.000204484979
17generate(0.809016279, 0.309016519, 0.500001451), (-0.309018627, -0.5, 0.809016371), (0.500000148, -0.809017176, -0.309016279)-81.9266201, 132.560299, 214.486495
18generate(-0.5, -0.809016693, -0.309017784), (0.809017498, -0.309017321, -0.499998983), (0.309015676, -0.500000286, 0.809017321)347.04665, 132.559842, 50.6335838
19generate(-0.5, 0.809017498, 0.309015676), (-0.809016693, -0.309017321, -0.500000286), (-0.309017784, -0.499998983, 0.809017321)50.6335225, 347.046627, 132.559927
20generate(0.809016518, -0.309017704, -0.500000333), (-0.309016761, 0.500000477, -0.809016789), (0.500000915, 0.809016429, 0.309016994)132.560201, 214.486464, -81.9266319
21generate(0.499998752, -0.809017909, -0.309016617), (-0.809017327, -0.309015747, -0.500000233), (0.309018142, 0.49999929, -0.809016994)214.486822, 347.046495, 132.559942
22generate(0.499998752, -0.809017327, 0.309018142), (-0.809017909, -0.309015747, 0.49999929), (-0.309016617, -0.500000233, -0.809016994)132.560057, 214.486636, 347.046566
23generate(0.809016518, -0.309016761, 0.500000915), (-0.309017704, 0.500000477, 0.809016429), (-0.500000333, -0.809016789, 0.309016994)-8.90124399E-5, 0.000105849606, 265.120017
24generate(0.309016994, -0.500000072, -0.80901695), (0.500001076, 0.809016556, -0.309016401), (0.809016329, -0.309018026, 0.500000439)265.120004, -0.000163124605, 0.000166771333
25generate(-0.809016994, -0.309016035, -0.500000593), (0.309018663, 0.499998852, -0.809017066), (0.499998968, -0.80901807, -0.309015846)347.046537, 132.55994, 214.486713
26generate(-0.809016994, 0.309018663, 0.499998968), (-0.309016035, 0.499998852, -0.80901807), (-0.500000593, -0.809017066, -0.309015846)132.559915, 214.486753, 347.046522
27generate(0.309016994, 0.500001076, 0.809016329), (-0.500000072, 0.809016556, -0.309018026), (-0.80901695, -0.309016401, 0.500000439)-81.92664, 132.560204, 214.486443
28generate(-0.499998958, 0.809017577, 0.309017155), (0.809017577, 0.309016151, 0.499999578), (0.309017155, 0.499999578, -0.809017193)50.6331777, -81.9264951, 132.560061
29generate(-0.809016672, -0.309017577, -0.500000161), (-0.309017577, -0.499998958, 0.809017416), (-0.500000161, 0.809017416, 0.30901563)347.046641, 132.559883, 50.6335607
30generate(0.80901721, 0.309016384, 0.500000028), (0.309017388, 0.499999784, -0.809016977), (-0.499999407, 0.809017361, 0.309016994)-81.926537, 132.559974, 50.6332872
31generate(0.500000566, 0.809016401, 0.309017632), (0.809017022, -0.30901756, -0.499999606), (-0.309016007, 0.50000061, -0.809016994)-81.9266664, 132.560019, 214.486374
32generate(0.500000566, 0.809017022, -0.309016007), (0.809016401, -0.30901756, 0.50000061), (0.309017632, -0.499999606, -0.809016994)-0.000209467224, 7.2711595E-5, 265.119863
33generate(0.80901721, 0.309017388, -0.499999407), (0.309016384, 0.499999784, 0.809017361), (0.500000028, -0.809016977, 0.309016994)50.633255, -81.9265246, 132.559994
34generate(0.500000367, -0.809016939, -0.309016545), (0.809016456, 0.309016994, 0.500000871), (-0.30901781, -0.500000089, 0.809016628)214.48647, -81.9266296, 132.560168
35generate(-0.309016035, -0.500000449, -0.809017083), (0.499999184, -0.809016994, 0.309018314), (-0.809017865, -0.309016268, 0.49999904)347.04653, 132.559933, 214.486732
36generate(-0.309016035, 0.499999184, -0.809017865), (-0.500000449, -0.809016994, -0.309016268), (-0.809017083, 0.309018314, 0.49999904)214.486682, 347.046549, 132.559964
37generate(0.500000367, 0.809016456, -0.30901781), (-0.809016939, 0.309016994, -0.500000089), (-0.309016545, 0.500000871, 0.809016628)0.000130840014, 265.120005, -0.000126332785
38generate(0.809017201, -0.309016746, -0.499999818), (0.309016908, -0.5, 0.809017028), (-0.499999719, -0.809017089, -0.309017201)132.559916, 50.6334216, 347.046588
39generate(0.809017201, 0.309016908, -0.499999719), (-0.309016746, -0.5, -0.809017089), (-0.499999818, 0.809017028, -0.309017201)50.6333613, 347.046565, 132.559999
40generate(1.24413229E-6, -1.40470472E-7, -1), (1, -1.38460104E-6, 1.24413248E-6), (-1.38460121E-6, -1, 1.4046875E-7)265.119854, 1.86207413E-5, 265.120165
41generate(1.24413229E-6, 1, -1.38460121E-6), (-1.40470472E-7, -1.38460104E-6, -1), (-1, 1.24413248E-6, 1.40468749E-7)1.86207903E-5, 265.120202, 265.119816
42generate(4.4686898E-7, -1, 5.0835724E-7), (-9.5522602E-7, 5.08356813E-7, 1), (-1, -4.46869465E-7, -9.55225792E-7)265.119873, 5.92370596E-5, 265.120186
43generate(4.4686898E-7, -9.5522602E-7, -1), (-1, 5.08356813E-7, -4.46869465E-7), (5.08357239E-7, 1, -9.55225793E-7)265.120067, 265.119992, 5.9236973E-5
44generate(-0.309016639, 0.500000087, -0.809017076), (0.500000707, 0.809016639, 0.30901678), (0.809016693, -0.309017784, -0.5)214.486538, -81.9266038, 132.560145
45generate(-0.309016639, 0.500000707, 0.809016693), (0.500000087, 0.809016639, -0.309017784), (-0.809017076, 0.30901678, -0.5)-0.000100893135, 0.000140224081, 265.120039
46generate(-0.80901711, 0.309016966, 0.499999831), (0.309016966, -0.49999979, 0.809017135), (0.499999831, 0.809017135, 0.3090169)132.560041, 50.6333718, -81.9265692
47generate(-2.19623925E-6, -1, -8.08350253E-7), (1.38789382E-6, 8.08347205E-7, -1), (1, -2.19624037E-6, 1.38789204E-6)265.120398, 265.119709, 0.000107155101
48generate(-2.19623925E-6, 1.38789382E-6, 1), (-1, 8.08347205E-7, -2.19624037E-6), (-8.08350252E-7, -1, 1.38789204E-6)0.000107154718, 265.120184, 265.119923
49generate(0.309017443, -0.500000871, -0.809016285), (-0.500000871, -0.809016395, 0.309017155), (-0.809016285, 0.309017155, -0.500001049)265.119962, 265.120015, 265.120024
50generate(-0.500002512, -0.809015586, 0.309016617), (-0.809015586, 0.309019013, -0.500001032), (0.309016617, -0.500001032, -0.809016501)265.120196, 265.119682, 265.120121
51generate(-0.309017538, -0.499999742, -0.809016946), (-0.499999043, 0.809017538, -0.30901712), (0.809017378, 0.309015989, -0.5)347.046617, 132.559818, 50.6334968
52generate(-0.309017538, -0.499999043, 0.809017378), (-0.499999742, 0.809017538, 0.309015989), (-0.809016946, -0.30901712, -0.5)132.559894, 50.6334414, 347.046596
53generate(-0.499999085, 0.809017399, -0.309017416), (0.809017399, 0.309015908, -0.500000017), (-0.309017416, -0.500000017, -0.809016823)132.559881, 50.6335071, 347.046621
54generate(-0.50000022, -0.809017155, -0.309016218), (-0.809017155, 0.30901809, 0.499999062), (-0.309016218, 0.499999062, -0.809017871)347.046533, 132.56, 214.486723
55generate(-1, 6.44E-7, -3.70229265E-13), (6.44E-7, 1, -1.1494E-6), (-3.69984335E-13, -1.1494E-6, -1)265.119915, 6.6995939E-5, 265.120152
56generate(0.30901506, -0.500000199, 0.80901761), (-0.500000199, -0.80901656, -0.309017809), (0.80901761, -0.309017809, -0.4999985)50.6336156, 347.046662, 132.559828
57generate(-0.809017924, -0.309016205, 0.499998983), (-0.309016205, -0.50000012, -0.809017222), (0.499998983, -0.809017222, 0.309018044)214.486739, 347.046527, 132.560026
58generate(1, -1.4256E-6, 7.37E-7), (-1.4256E-6, -1, -5.25456065E-13), (7.37E-7, -5.25211135E-13, -1)9.12809867E-5, 265.120189, 265.119902
59generate(-0.80901623, 0.309017183, -0.500001121), (0.309017183, -0.500001392, -0.809016062), (-0.500001121, -0.809016062, 0.309017621)265.120022, 265.120036, 265.119942
60generate(0.309017839, 0.49999935, -0.809017073), (0.49999935, -0.809017614, -0.309016423), (-0.809017073, -0.309016423, -0.500000225)132.559985, 214.486678, 347.04655

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Components

#1: Protein 6,7-dimethyl-8-ribityllumazine synthase / Lumazine synthase / DMRL synthase / LS / Lumazine synthase


Mass: 17769.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus (bacteria) / Gene: ribH, aq_132 / Production host: Escherichia coli (E. coli)
References: UniProt: O66529, 6,7-dimethyl-8-ribityllumazine synthase

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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: lumazine synthase / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Aquifex aeolicus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / 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 FIELDBright-field microscopy / 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: I (icosahedral)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8565 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
Atomic model buildingPDB-ID: 1hqk

1hqk


Accession code: 1hqk / Source name: PDB / Type: experimental model

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