Table 1 Comparison of experimentally obtained RDA values and those obtained from AV simulations

From: Direct observation of subunit rotation during DNA strand exchange by serine recombinases

SR

State

D/A

E

R0

RDA (Å)

E (AV/ γδ)

<RDA>(Å) (AV/ γδ)

Sin Q115R (37 °C)

lnr

Atto532/ Cy5

0.16 (0.04)

67.4

88.9 (4.5)

0.13 (0.02)

92.4 (2.1)

Sin Q115R (37 °C)

cnr

Atto532/ Cy5

0.29 (0.02)

67.4

78.2 (1.3)

0.31 (0.05)

77.0 (1.6)

Sin Q115R (37 °C)

cr

Atto532/ Cy5

0.50 (0.03)

67.4

67.4 (1.3)

0.66 (0.08)

60.2 (1.8)

Sin Q115R (37 °C)

lr

Atto532/ Cy5

0.77 (0.04)

67.4

55.1 (2.1)

0.87 (0.05)

49.3 (1.7)

Sin Q115R (37 °C, 20 mM Mg)

lnr

Atto532/ Cy5

0.13 (0.04)

67.4

92.5 (5.7)

0.13 (0.03)

92.4 (2.1)

Sin Q115R (37 °C, 20 mM Mg)

cnr

Atto532/ Cy5

0.29 (0.03)

67.4

78.2 (2.5)

0.31 (0.05)

77.0 (1.6)

Sin Q115R (37 °C, 20 mM Mg)

cr

Atto532/ Cy5

0.53 (0.03)

67.4

66.1 (1.3)

0.66 (0.08)

60.2 (1.8)

Sin Q115R (37 °C, 20 mM Mg)

lr

Atto532/ Cy5

0.79 (0.04)

67.4

54.6 (2.2)

0.87 (0.05)

49.3 (1.7)

Sin Q115R (21 °C)

lnr

Atto532/ Cy5

0.17 (0.02)

67.4

87.8 (4.2)

0.13 (0.03)

92.4 (2.1)

Sin Q115R (21 °C)

cnr

Atto532/ Cy5

0.34 (0.02)

67.4

75.3 (2.2)

0.31 (0.05)

77.0 (1.6)

Sin Q115R (21 °C)

cr

Atto532/ Cy5

0.54 (0.05)

67.4

65.6 (4.4)

0.66 (0.08)

60.2 (1.8)

Sin Q115R (21 °C)

lr

Atto532/ Cy5

0.78 (0.03)

67.4

54.6 (3.2)

0.87 (0.05)

49.3 (1.7)

Tn3 NM

lnr

Cy3B/ Cy5

0.12 (0.03)

67.0

93.4 (4.5)

0.22 (0.03)

82.5 (2.3)

Tn3 NM

cnr

Cy3B/ Cy5

0.31 (0.03)

67.0

76.6 (1.8)

0.40 (0.08)

71.4 (1.7)

Tn3 NM

cr

Cy3B/ Cy5

0.54 (0.03)

67.0

65.2 (1.3)

0.52 (0.10)

66.7 (2.3)

Tn3 NM

lr

Cy3B/ Cy5

0.73 (0.02)

67.0

56.7 (1.0)

0.79 (0.06)

53.7 (1.8)

Sin Q115R (MFD)

lnr

Alexa488/ Cy5

–

51.4

–

0.03 (0.01)

92.4 (2.1)

Sin Q115R (MFD)

cnr

Alexa488/ Cy5

–

51.4

–

0.09 (0.01)

75.8 (1.6)

Sin Q115R (MFD)

cr

Alexa488/ Cy5

–

51.4

–

0.33 (0.04)

57.8 (1.6)

Sin Q115R (MFD)

lr

Alexa488/ Cy5

0.50 (0.02)

51.4

52.8 (0.7)

0.63 (0.04)

47.2 (1.8)

Sin Q115R (20 mM Mg) (MFD)

lr

Alexa488/ Cy5

0.50 (0.02)

51.4

52.8 (0.7)

0.63 (0.04)

47.2 (1.8)

Tn3 NM-S10A (inversion) (MFD)

lnr

Alexa488/ Cy5

0.23 (0.01)

51.4

62.9 (0.6)

0.06 (0.01)

80.7 (2.3)

Tn3 NM-S10A (deletion) (MFD)

lnr

Alexa488/ Cy5

0.23 (0.01)

51.4

62.9 (0.6)

0.10 (0.01)

74.1 (2.3)

Tn3 NM (MFD)

cnr

Alexa488/ Cy5

0.24 (0.01)

51.4

62.3 (0.6)

0.14 (0.02)

66.4 (1.6)

Tn3 NM (MFD)

cr

Alexa488/ Cy5

0.37 (0.01)

51.4

56.2 (0.4)

0.24 (0.05)

62.1 (2.2)

Tn3 NM (MFD)

lnr

Alexa488/ Cy5

0.5 (0.01)

51.4

51.4 (0.3)

0.50 (0.05)

51.9 (1.7)

  1. Donor-acceptor distances (RDA) for SUL25/Sin Q115R complexes in the presence and absence of Mg2+, and UL25/Tn3 NM complexes with no Mg2+, were calculated using the Förster equation using values of E obtained from HaMMy (TIRF) or Gaussian fitting (MFD). The Forster radii used were, for Atto 532/ Cy5, R0 = 67.4 Å50,51; for Alexa 488/ Cy5, R0 = 51.4 Å52,53; and for Cy3B/ Cy5, R0 = 67 Å54. <RDA>E is the FRET-averaged distance between the dyes, resulting from integrating overall possible FRET efficiencies determined through AV simulation of various fluorophore positions modelled on crystal structures of γδ-resolvase7,9. Standard deviations (given in parentheses) in experimental values of E were determined through clustering analysis of the transition density plots in MASH-FRET34 or were determined by AV simulation.
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