Does the pcie slot matter for performance? Which card where?

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Hello, still learning about mining and setups.
I have a MSI B450 that has 2 16x pcie slots and 3 1x pcie slots
My cards are a 3080TI, RX6600, and 2 RX580’s currently.
I have Above 4G memory/ Crypto Currency mining turned on which I think makes all the lanes 4x?

So does it matter which card goes where? Will it affect performance? What would you all recommend?
Thanks.

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