native-MS, crosslinking MS, HDX-MS). Интегральный подход и моделирование белков по
гомологии (iTasser). Примеры.Случанко Н.Н.
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Лекция 4+5 Другие методы исследования структуры белков (SAXS/SANS, Cryo-EM,
Содержание
- 1. Лекция 4+5 Другие методы исследования структуры белков (SAXS/SANS, Cryo-EM,
- 2. Small-angle X-ray scattering (SAXS) + Small-angle neutron scattering (SANS) II Small-angle scattering (SAS)
- 3. SAXS popularityBlanchet C. (c)
- 4. Основы SAS~1 photon in 106 incident photons
- 5. Основы SASs and q are just alternative
- 6. Contrast and careful buffer subtractionMeasured in the
- 7. Kikhney A (c)Processed final curve !
- 8. ОсобенностиМакромолекулы свободно вращаются, не ориентированы строго при
- 9. Слайд 9
- 10. https://www.embl-hamburg.de/biosaxs/software.html
- 11. Форма кривой SAXS сильно зависит от размера и формы частиц
- 12. What does the curve already tell us
- 13. Pairwise distance distribution function p(r)Blanchet C. (c)FFT
- 14. Pairwise distance distribution function p(r)Blanchet C. (c)FFTDmaxmaximum intra-particle distance
- 15. Слайд 15
- 16. Kikhney A (c)DAMMIF programhttps://www.embl-hamburg.de/biosaxs/dammif.html
- 17. Слайд 17
- 18. Слайд 18
- 19. Слайд 19
- 20. Linear ≠ monodisperse(also for mixed systems)
- 21. Guinier plot and RgAverage of square center-of-mass
- 22. Kratky plot and flexibilityIdentification of unfolded samplesGlobular proteins have bell-shaped curves (parabola)
- 23. If X-ray structures are available…Atomistic modeling:Validation of
- 24. Validation of the crystal structure in solution situation1.75A
- 25. Comparison of the crystal structures and ab initio envelopes
- 26. Conformational change
- 27. Conformational changeFRP dimer
- 28. SEC-SAXS for contaminated samplesM. Graewert (c)
- 29. SASBDB https://www.sasbdb.org/aboutSASBDB/
- 30. Трезвый взгляд на SAXSДает хорошую информацию
- 31. SANSNeutron source (rare)Non-ionizing radiationCoherent scattering (=elastic)Incoherent scattering
- 32. Samples for SAXS and SANS
- 33. CryoEMhttps://www.nature.com/news/the-revolution-will-not-be-crystallized-a-new-method-sweeps-through-structural-biology-1.18335 https://www.youtube.com/watch?v=aHhmnxD6RCI
- 34. Resolution revolutionпоявление прямых детекторов электроновразвитие софта для обработки огромного количества картиноксовершенствование микроскопов, адаптация к криоусловиям
- 35. The recipe includeshttps://www.youtube.com/watch?v=BJKkC0W-6Qk
- 36. The process of Cryo-EM single particle analysis techniqueby cross-correlation
- 37. Features, 2D->3DBiological samples – low doses and
- 38. Contrast transfer function and defocusAt perfect focus,
- 39. Contrast transfer function and defocusAt perfect focus,
- 40. Contrast transfer function and defocusDOI: 10.1142/9781848164666_0001
- 41. Single particle cryoEM requires tons of imagesParticle
- 42. Signal and noise5:11:1S/N = 1:1 (0 dB)Improving
- 43. Einstein from noiseAn image of Einstein appears
- 44. Обучение криоЭМhttps://ru.coursera.org/learn/cryo-emhttps://em-learning.com https://www.youtube.com/watch?v=Bk5lBvwSe-s Prof. Yifan Cheng
- 45. Слайд 45
- 46. Cryo-electrotomography (Cryo-ET)
- 47. Cryo-electrotomography (Cryo-ET)https://doi.org/10.1371/journal.pbio.3000050
- 48. NMR – nuclear magnetic resonancehttps://www.youtube.com/watch?v=0s7Cbl8bZLMhttps://www.youtube.com/watch?v=eY0NyE0SQjE NMR made super easy:
- 49. The output of the (successful) multidimensional NMR
- 50. NMRSpin up OrSpin downhttps://www.youtube.com/watch?v=PmYwYUQw-Rw
- 51. Properties of some nucleiBonvin A (c)
- 52. NMR sampleBonvin A (c)
- 53. Bonvin A (c)Nuclear spinЧастота прецессии (Ларморова частота)
- 54. Energy between α (+1/2) and β (-1/2) levels1H
- 55. Transitions between levels are possible
- 56. NMR, a spectroscopy techniqueIn a magnetic field magnetic nuclei will resonate with a specific frequency
- 57. Bonvin A (c)
- 58. Magnetization (M) gets back to the B0-oriented
- 59. Chemical shift due to the local environment
- 60. The local electronic environment of the nucleus may change the frequency: shielding effectresonancesdeshieldingshielding
- 61. Pulse method to deliver a set of ν and then do …Good old Fourier !
- 62. 1D 1H-spectrum of ethanolCH3–CH2–OH(CH3)4SiChemical shiftSeveral peaks due to spin-spin interaction
- 63. 2D spectraSeries of pulses to cause transitions2D
- 64. Спектр 15N-1H HSQC apo-CTDH (0.5 mM), при
- 65. Resolution of the peaks is increased upon increasing dimensionality
- 66. Structural models of small proteinsDistances between neighboring atomsAngles ψ and φ of the polypeptide chain2MOU.pdbSTARD620 structures
- 67. NMR tackles both structured proteins and IDPs
- 68. NMR tackles both structured proteins and IDPs
- 69. i-Tasser. Protein structure predictionFASTA format of sequencehttps://zhanglab.ccmb.med.umich.edu/I-TASSER/
- 70. Comparison of different structural techniques
- 71. Integrated approaches in structural biologyX-ray crystallographySAXSNMRCryoEMAuxillary techniques:
- 72. Native-MShttps://doi.org/10.3389/fmicb.2018.01397
- 73. Native-MShttps://doi.org/10.1007/s13361-018-2061-4highly charged complexesno additional chargeshttps://www.nature.com/articles/nmeth.1265 https://www.pnas.org/content/116/4/1116 DOI: 10.1007/978-1-4939-7151-0_11
- 74. Hydrogen/deuterium exchange mass-spectrometryYoshitomo Hamuro ©0°C, H+https://doi.org/10.1016/j.sbi.2019.06.007https://onlinelibrary.wiley.com/doi/abs/10.1002/pro.3790 amide protons
- 75. Pseudoatomic models built by a combination of:Single particle Cryo-EMCrosslinking MSHDX MSModelling
- 76. Cryo-EM micrograph of human alphaA-crystallin12-mer16-mer20-mer
- 77. Cryo-EM 3D reconstructions of human αA-crystallin (reduced) oligomers Scale bar, 10 nm239 kDa319 kDa398 kDa
- 78. Crosslinking by BS3 and MSFragmentation spectrum of
- 79. Pseudoatomic model of the 16-merModelling by molecular
- 80. Effect of alphaA-crystallin oxidationFar-UV CDNear-UV CDanSECAUC-SV14S25S50nm50nmNegative stain TEM
- 81. HDX-MS shows incresed local structural dynamics of
- 82. Скачать презентанцию
Small-angle X-ray scattering (SAXS) + Small-angle neutron scattering (SANS) II Small-angle scattering (SAS)
Слайды и текст этой презентации
Слайд 1Лекция 4+5 Другие методы исследования структуры белков (SAXS/SANS, Cryo-EM, Cryo-electrotomography, NMR,
Слайд 2Small-angle X-ray scattering (SAXS)
+
Small-angle neutron scattering (SANS)
II
Small-angle scattering (SAS)
Слайд 5Основы SAS
s and q are just alternative designations of the
scattering vector,
usually from 0 to 0.5 Å-1
~ 10-20 Å
Слайд 6Contrast and careful buffer subtraction
Measured in the same cell, buffer
exactly matches
Difference in the scattering density (contrast):
Слайд 8Особенности
Макромолекулы свободно вращаются, не ориентированы строго при падающем пучке X-ray
Может
быть несколько конформаций одновременно
В результате наблюдаемое рассеяние это сферическое усреднение
(изотропное) и усреднение по времениТеряется 3D информация
Данные при радиальном усреднении дают 1D кривую распределения I(q) с небольшим числом параметров
Данные – 1D кривая…
Слайд 12What does the curve already tell us about the size
of the particles? What is the resolution?
s, nm-1
logI
d ~ 1.4
nm
Слайд 14Pairwise distance distribution function p(r)
Blanchet C. (c)
FFT
Dmax
maximum intra-particle distance
Слайд 21Guinier plot and Rg
Average of square center-of-mass distances in the
molecule
Measure of the overall size of the molecule
R
A. Guinier
Слайд 22Kratky plot and flexibility
Identification of unfolded samples
Globular proteins have bell-shaped
curves (parabola)
Слайд 23If X-ray structures are available…
Atomistic modeling:
Validation of the crystal structure
against solution situation
Rigid-body fitting
Missing fragments (loops)
Conformational transitions
Theoretical SAXS profile can
be calculated by CRYSOL program, necessary for fitting
Слайд 30 Трезвый взгляд на SAXS
Дает хорошую информацию о гидродинамических свойствах
частиц (структурных свойствах) в растворе
Хорош для тестирования гипотез о структуре,
форме, комплексе и т.п.Вспомогательный метод структурной биологии
Необходимо сверяться с как можно большим количеством экспериментальных данных (стехиометрия, олигомерное состояние, размеры, масса, радиус, пространственные ограничения, знания об интерфейсах, топологии субъединиц и т.п.)
В одиночку SAXS не стоит использовать для структурной биологии (ambiguity)
Слайд 31SANS
Neutron source (rare)
Non-ionizing radiation
Coherent scattering (=elastic)
Incoherent scattering (1H affects)
Contrast is
very different in H2O and D2O
SAXS and SANS are complementary!
Contrast
variation by increasing D2O content:Difference in the scattering density (contrast)
Study of conformational changes of selected proteins within the complexes !!!
Features:
Слайд 33CryoEM
https://www.nature.com/news/the-revolution-will-not-be-crystallized-a-new-method-sweeps-through-structural-biology-1.18335
https://www.youtube.com/watch?v=aHhmnxD6RCI
Слайд 34Resolution revolution
появление прямых детекторов электронов
развитие софта для обработки огромного количества
картинок
совершенствование микроскопов, адаптация к криоусловиям
Слайд 37Features, 2D->3D
Biological samples – low doses and dehydration (high vacuum)
Freezing
allows to avoid these, but the images have a very
low contrastEach picture - 2D projection of a 3D object
Multiple 2D projections can be used to reconstruct the 3D object
DOI:
10.1142/9781848164666_0001
http://www.ejectamenta.com/Imaging-Experiments/fourierimagefiltering.html
Слайд 38Contrast transfer function and defocus
At perfect focus, biological specimens produce
little contrast in vitreous ice.
To produce phase contrast, pictures are
taken underfocus, at the expense of systematic alteration of the image data (not all waves are well transferred -> CTF)Each picture is undergoing FT to see Thon rings (~resolution rings in Xtallography) – contrast transfer function (CTF)
Some waves are lost but can be CTF-corrected upon changing defocus (d below)
Слайд 39Contrast transfer function and defocus
At perfect focus, biological specimens produce
little contrast in vitreous ice.
To produce phase contrast, pictures are
taken underfocus, at the expense of systematic alteration of the image data (not all waves are well transferred -> CTF)Each picture is undergoing FT to see Thon rings (~resolution rings in Xtallography) – contrast transfer function (CTF)
Some waves are lost but can be CTF-corrected upon changing defocus (d below)
d is varied
Слайд 41Single particle cryoEM requires tons of images
Particle orientations are classified
by cross-correlation
Each class should be represented by thousands of images
Also,
at different defocus valuesSome images are discarded
Слайд 42Signal and noise
5:1
1:1
S/N = 1:1 (0 dB)
Improving S/N by repetition
and averaging
4 measurements = 2 *S/N
50S ribosome projection
Accurate alignment and
the target model are important
Слайд 43Einstein from noise
An image of Einstein appears from averaged 1000
images of pure white noise by using a normalized cross-correlation
function and the photo as a model.doi: 10.1016/j.jsb.2008.12.008
Слайд 44Обучение криоЭМ
https://ru.coursera.org/learn/cryo-em
https://em-learning.com
https://www.youtube.com/watch?v=Bk5lBvwSe-s
Prof. Yifan Cheng
Слайд 48NMR – nuclear magnetic resonance
https://www.youtube.com/watch?v=0s7Cbl8bZLM
https://www.youtube.com/watch?v=eY0NyE0SQjE
NMR made super easy:
Слайд 49The output of the (successful) multidimensional NMR experiment
A set of
structural models that satisfy the experimental constraints but also obey
the chemistry rules
Слайд 56NMR, a spectroscopy technique
In a magnetic field magnetic nuclei will
resonate with a specific frequency
Слайд 58Magnetization (M) gets back to the B0-oriented position after being
affected by external field
B0
Exponential decay
Free induction decay (FID)
Relaxation
=спад свободной индукции
Слайд 59Chemical shift due to the local environment changing frequency of
the nuclei
Expressed as part per million (ppm) by comparison to
the reference frequency:(may also be presented in Hz)
Слайд 60The local electronic environment of the nucleus may change the
frequency: shielding effect
resonances
deshielding
shielding
Слайд 621D 1H-spectrum of ethanol
CH3–CH2–OH
(CH3)4Si
Chemical shift
Several peaks due to spin-spin interaction
Слайд 632D spectra
Series of pulses to cause transitions
2D Fourier transformation
Proximal functional
groups affect the magnetization of a particular nucleus in the
structure
Слайд 64Спектр 15N-1H HSQC apo-CTDH (0.5 mM), при 800 MHz и
35°С. Отнесены сигналы амидных групп белковой цепи.
ApoCTDH
6FEJ.pdb
http://pdbflex.org/index.html
Наложение спектров 15N-1H
HSQC apo-CTDH (красные) и CTDH-Canthaxanthin (синие)K14
A17
L31
P36
G56
G59
L67
G97
V108
F112
H122
Слайд 66Structural models of small proteins
Distances between neighboring atoms
Angles ψ and
φ of the polypeptide chain
2MOU.pdb
STARD6
20 structures
Слайд 69i-Tasser. Protein structure prediction
FASTA format of sequence
https://zhanglab.ccmb.med.umich.edu/I-TASSER/
Слайд 71Integrated approaches in structural biology
X-ray crystallography
SAXS
NMR
CryoEM
Auxillary techniques: fluorescence resonanse energy
transfer (FRET), limited proteolysis, native-MS, crosslinking, HDX, molecular dynamics and
computational biology
Слайд 73Native-MS
https://doi.org/10.1007/s13361-018-2061-4
highly charged complexes
no additional charges
https://www.nature.com/articles/nmeth.1265
https://www.pnas.org/content/116/4/1116
DOI: 10.1007/978-1-4939-7151-0_11
Слайд 74Hydrogen/deuterium exchange mass-spectrometry
Yoshitomo Hamuro ©
0°C, H+
https://doi.org/10.1016/j.sbi.2019.06.007
https://onlinelibrary.wiley.com/doi/abs/10.1002/pro.3790
amide protons
Слайд 75Pseudoatomic models built by a combination of:
Single particle Cryo-EM
Crosslinking MS
HDX
MS
Modelling
Слайд 77Cryo-EM 3D reconstructions of human αA-crystallin (reduced) oligomers
Scale bar,
10 nm
239 kDa
319 kDa
398 kDa
Слайд 78Crosslinking by BS3 and MS
Fragmentation spectrum of a cross-linked peptide
with an intramolecular link between K70 and K99
Fragmentation spectrum
of a cross-linkedpeptide with an intermolecular cross-link between M1 and M1
bis(sulfosuccinimidyl)suberate (BS3)
11.4A
Слайд 79Pseudoatomic model of the 16-mer
Modelling by molecular dynamics flexible fitting
was based on:
-shape, symmetry and low-resolution features from 9-10 Å
resolution Cryo-EM maps-crystal structures of truncated versions (domains)
-crosslinking MS data (pairs of residues located within certain distance)
-stereochemistry restraints
Слайд 80Effect of alphaA-crystallin oxidation
Far-UV CD
Near-UV CD
anSEC
AUC-SV
14S
25S
50nm
50nm
Negative stain TEM
Слайд 81HDX-MS shows incresed local structural dynamics of alphaA-crystallin
Deuteration uptake behavior
of the oxidized and reduced αA
Difference in local relative deuterium
uptake (ΔD uptake αAox − αAred)
